Dermal filler compositions

ABSTRACT

The present invention provides highly injectable, long-lasting hyaluronic acid-based hydrogel dermal filler compositions made with a di-amine or multiamine crosslinker in the presence of a carbodiimide coupling agent.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/568,618, filed Dec. 8, 2011, and is a continuation-in-part ofU.S. patent application Ser. No. 13/615,193, filed on Sep. 13, 2012,which claims priority to U.S. Provisional Patent Application No.61/534,780, filed on Sep. 14, 2011, and which is a continuation-in-partof U.S. patent application Ser. No. 13/593,313, filed on Aug. 23, 2012,which claims priority to and the benefit of U.S. Provisional PatentApplication No. 61/527,335 filed on Aug. 25, 2011 and which is acontinuation-in-part of U.S. patent application Ser. No. 13/486,754,filed on Jun. 1, 2012, which claims priority to U.S. Provisional PatentApplication No. 61/493,309, filed on Jun. 3, 2011, the entire content ofeach of these documents being incorporated herein by this specificreference.

BACKGROUND

The present invention generally relates to dermal filler compositions,for example, but not limited to, dermal filler compositions that areeffective for treatment of fine lines in skin.

Skin aging is a progressive phenomenon, occurs over time and can beaffected by lifestyle factors, such as alcohol consumption, tobacco andsun exposure. Aging of the facial skin can be characterized by atrophy,slackening, and fattening. Atrophy corresponds to a massive reduction ofthe thickness of skin tissue. Slackening of the subcutaneous tissuesleads to an excess of skin and ptosis and leads to the appearance ofdrooping cheeks and eye lids. Fattening refers to an increase in excessweight by swelling of the bottom of the face and neck. These changes aretypically associated with dryness, loss of elasticity, and roughtexture.

Hyaluronic acid (HA), also known as hyaluronan, is a non-sulfatedglycosaminoglycan that is distributed widely throughout the human bodyin connective, epithelial, and neural tissues. Hyaluronic acid isabundant in the different layers of the skin, where it has multiplefunctions such as, e.g., to ensure good hydration, to assist in theorganization of the extracellular matrix, to act as a filler material;and to participate in tissue repair mechanisms. However, with age, thequantity of hyaluronic acid, collagen, elastin, and other matrixpolymers present in the skin decreases. For example, repeated exposed toultra violet light, e.g., from the sun, causes dermal cells to bothdecrease their production of hyaluronan as well as increase the rate ofits degradation. This loss of materials results in various skinconditions such as, e.g., wrinkling, hollowness, loss of moisture andother undesirable conditions that contribute to the appearance of agingInjectable dermal fillers have been successfully used in treating theaging skin. The fillers can replace lost endogenous matrix polymers, orenhance/facilitate the function of existing matrix polymers, in order totreat these skin conditions.

In humans, the residence time of un modified hyaluronic acid is a fewdays, as the polymer chains are easily degraded by enzymes and freeradicals found in the body. Commercially available dermal fillers aregenerally prepared by the crosslinking of hydroxyl groups of HA achemical crosslinker. Commercial dermal filler gels are available whichcontain hyaluronic acid synthesized with divinyl sulfone (DVS),1,4-butanediol diglycidyl ether (BDDE), or 1,2,7,8-diepoxyoctane (DEO).The softness and tissue augmentation effect of dermal fillers can becontrolled by changing the cross-linking density with variouscrosslinkers.

To improve on the residence time, the linear chains of hyaluronic acidare typically crosslinked with a small molecular crosslinker like BDDE.Crosslinking is performed at very high pH (>12) and at temperatures ofabout 50° C. It has been reported that the degradation rate constant ofHA is increased roughly 100 times when the temperature and pH are bothincreased from 40 to 60° C. and 7 to 11 respectively. This drawback hasled to the quest for new crosslinkers and crosslinking chemistries forHA that would involve milder conditions.

In the present invention, crosslinking HA is crosslinked with a couplingagent and the use of small multiamine crosslinkers, which form amidebonds with the carboxylic functional groups of HA chains. In the presentinvention, crosslinking may be done at low pH levels, for example, at apH between 4-7 and temperatures between 20 and 37° C., conditions atwhich degradation of HA is minimal.

Bioconjugate Chemistry, 2010, 21, 240-247: Joem Y., et al., Effect ofcross-linking reagents for hyaluronic acid hydrogel dermal fillers ontissue augmentation and regeneration discusses the use of HMDA toprepare a cross linked HA dermal filler for tissue augmentation. Thispublication is incorporated herein in its entirety by this specificreference.

Tyndall effect is an adverse event occurring in some patientsadministered with hyaluronic acid (HA)-based dermal fillers. Tyndalleffect is characterized by the appearance of a blue discoloration at theskin site where a dermal filler had been injected, which representsvisible hyaluronic acid seen through the translucent epidermis. Clinicalreports suggest that filler administration technique and skin propertiescan influence the manifestation of this adverse event. Fillers with highstiffness and elasticity are successfully used to correct areas on theface like nasolabial folds, cheeks, and chin without any fear of facialdiscoloration, as the materials are injected in the mid and deep dermisregions. However, when these filler materials are used to correctsuperficial, fine line wrinkles, for example, tear trough, glabellarlines periorbital lines, smile lines, or forehead, or mistakenly appliedtoo superficially in the upper regions of the dermis, a bluishdiscoloration of the skin is often observed. This phenomenon, which isthought to be the result of Tyndall effect, leaves a semi-permanentdiscoloration of the application sites, and sometimes disappears onlyafter the administration of hyaluronidase to degrade the fillermaterial. Consequently, Tyndall effect is more common in patientstreated for superficial fine line wrinkles. Prolonged manifestation ofTyndall effect, typically for several months as long as the gel lasts inthe skin, is a cause of major concern among patients.

HA-based dermal filler gels have been specifically formulated to treat“fine line” wrinkles found around the tear trough, forehead, periobital,glabellar lines, etc. Many show Tyndall effect when injected toosuperficially. Though these gels are formulated to have low elasticmoduli by lightly crosslinking the linear HA chains with a small amountof BDDE and by reducing the final HA gel concentration, most of thecommercially available fine line gels still show tyndall when injectedsuperficially, and unfortunately, in vivo duration of these gels isquite marginal.

There is still a need for better dermal fillers for treating andimproving the appearance of aging skin.

SUMMARY

The present invention describes dermal filler compositions andformulation methods for preparing HA-based dermal fillers using newcrosslinking chemistries. Many of the presently described filler gels ofthe invention have been found to last significantly longer in vivo thancurrent commercially available gels. In some aspects of the invention,the compositions can be administered in the upper dermis withoutproducing any bluish discoloration of the skin, or at least nosignificant or noticeable bluish discoloration. In some aspects of theinvention, the dermal filler compositions are optically transparent andcan be used to enhance the appearance of the skin, e.g. add volume,fullness and reduce wrinkles and fine lines, without causing the bluediscoloration known as “tyndalling, which is sometimes associated withconventional optically transparent dermal fillers.

In one aspect of the present invention, long lasting, therapeutic dermalfiller compositions are provided which generally comprise abiocompatible polymer, for example, a hyaluronic acid componentcrosslinked with a di-amine or multiamine crosslinker.

In another aspect, such compositions are substantially opticallytransparent, and exhibit reduced or no perceptible blue discolorationwhen administered into a dermal region of a patient.

Methods of making an injectable dermal filler composition are alsoprovided. In one embodiment, the method comprises the steps ofcrosslinking hyaluronic acid (HA) with a multiamine crosslinker with theaid of a carbodiimide coupling agent.

In one aspect, the crosslinker is made up of at least three and a mosteight PEG chains emanating from a central point, each chain having aterminal amine group.

The crosslinker may contain PEG chains, for example, PEG chains having aleast one ethylene glycol unit and not more than 55 ethylene glycolunits.

In one aspect, the crosslinker is lysine methyl ester.

In another aspect, the crosslinker is 3-[3-(3-aminopropoxy)-2,2-bis(3-amino-propoxymethyl)-propoxy]-propylamine (4 AA) andis present at a concentration between about 10 μM to about 50 μM.

In one aspect, the HA is crosslinked in conjunction with a carbodiimidecoupling agent, for example, a water soluble coupling agent, forexample, a water soluble carbodiimide.

In another embodiment, the coupling agent is1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), andis present at a concentration between about 20 μM to 100 μM.

The coupling agent may be used in conjunction with a water solubleactivating agent, for example, N-hydroxysuccinimide (NHS) orN-hydroxysulfosuccinimide (sulfoNHS), present at a concentration betweenabout 5 μM to about 25 μM.

In one embodiment, the polymer is a polysaccharide, for example,hyaluronic acid. The hyaluronic acid includes a crosslinked componentand may further include a non-crosslinked component. The additive maycomprise a vitamin, for example, vitamin C, for example, a stabilizedform of vitamin C, or a vitamin C derivative, for example, L-ascorbicacid 2-glucoside (AA2G), ascobyl 3-aminopropyl phosphate (Vitagen) orsodium ascorbyl phosphate (AA2P).

In one aspect of the invention, the additive is a vitamin derivativewhich is covalently conjugated to the polymer by a suitable reactionprocess, for example, etherification, amidization or estherification.

The composition may be substantially optically transparent. Thecompositions. generally have a G′ value of between about 20 Pa and about150 Pa, for example, no greater than about 150 Pa and, for example, noless than about 20 Pa.

In another aspect of the invention, methods of treating fine lines inthe skin of a patient are provided. In one embodiment, the methodcomprises the steps of introducing, into skin of a patient, acomposition comprising a hyaluronic acid component crosslinked with adi-amine or multiamine crosslinker; and a carbodiimide coupling agent.The composition may be substantially optically transparent, and exhibitsreduced or no perceptible blue discoloration when administered into adermal region of a patient.

In another aspect of the invention, methods of improving aestheticappearance of a face are provided, the methods generally comprising thesteps of administering, to a dermal region of a patient, a substantiallyoptically transparent dermal filler composition that exhibits no orinsignificant Tyndall effect. The composition may be made by the stepsof crosslinking hyaluronic acid (HA) with a di-amine or multiaminecrosslinker in the presence of a carbodiimide coupling agent.

In yet another aspect of the invention, methods of reducing appearanceof fine lines in thin skin regions of a patient are provided, whereinthe method generally comprises administering to the patient a dermalfiller composition, at a depth of no greater than about 1 mm, asubstantially optically transparent hyaluronic acid based dermal fillercomposition comprising a hyaluronic acid component crosslinked with adi-amine or multiamine crosslinker.

In some embodiments, the composition is injected superficially, that is,at a depth of a depth of no greater than about 0.8 mm, no greater thanabout 0.6 mm, or no greater than about 0.4 mm.

In yet another aspect of the invention, a dermal filler composition isprovided which is generally comprises a hyaluronic acid componentcrosslinked with a di-amine or multiamine crosslinker in the presence ofa carbodiimide coupling agent. The composition may have a hyaluronicacid concentration of between about 14 mg/ml and about 30 mg/ml. Thehyaluronic acid may be a low molecular weight hyaluronic acid, forexample, a hyaluronic acid having a mean molecular weight of less thanabout 100K Da, less than 800 KDa, less than about 600 KDa, or less thanabout 400 KDa. These compositions may be especially useful and effectivein treating folds and wrinkles, including fine lines or superficialcreases in the skin, for example, even in very thin skin, for example,skin having a thickness of no greater than about 1 mm. In someembodiments, the compositions of the invention last at least 3 months,at least 6 months or up to a year after being introduced into the skin.

These and other aspects and advantages of the present invention may bemore readily understood and appreciated with referenced to the followingdrawings and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows images of skin of a mammal having injected therein acomposition in accordance with one aspect of the invention and twocommercial dermal fillers.

FIG. 2 shows a bar chart illustrating longevity in vivo of compositionsin accordance with one aspect of the invention, compared to a commercialdermal filler.

DETAILED DESCRIPTION

Hyaluronic acid (HA) is a naturally occurring glucosaminoglycan used toformulate dermal fillers that are used mainly for wrinkle reduction andvolumizing of the face. In humans, the residence time of unmodifiedhyaluronic acid is a few days, as the polymer chains are easily degradedby enzymes and free radicals found in the body. To improve on theresidence time, the linear chains of hyaluronic acid are usuallycrosslinked with a small molecular crosslinker like BDDE.

The present disclosure relates, in part to dermal fillers comprisingcrosslinked HA small di-amine and multiamine crosslinkers, which formamide bonds with the carboxylic functional groups of HA chains. In idealconditions, EDC activates the carboxylic acid groups of HA, and theactivated carboxylic acid groups then react with the amines. In oneaspect, crosslinking is done at pH between about 4 and about 7 and attemperatures between about 20° C. and about 37° C. Under theseconditions, it has been discovered that degradation of HA is minimal.

Linear diamine crosslinkers like hexamethylene diamine (HMDA), lysine,lysine methyl ester, have been used to crosslink HA for variousapplications. The problem with crosslinking HA via EDC chemistry is theconcurrent formation of ester bonds between carboxylic acid groups andthe hydroxyl groups of HA even in the presence of these multiamines.

Ester bonds are very labile, and are easily hydrolyzed at hightemperatures. HA hydrogels made by ester crosslinking are generally notrobust and cannot be sterilized with moist steam.

It has been a challenge to minimize the formation of ester bonds duringEDC mediated crosslinking of HA by amines. In one approach, a highNH₂:HA feed ratio is used, and addition of the amine to the hydrated HAis done before addition of EDC. This seems to increase the number ofamide bonds formed with respect to the number of ester bonds. It isbelieved that since amine groups are better nucleophiles than hydroxylgroups, more amide bonds are expected to be formed. Another approach isperforming the crosslinking at high pH in order to minimize ester bondformation, as these bonds are less stable at higher pH. One other methodis to use a very low HA reaction concentration, for example, at betweenabout 1% and about 5 wt %. At such low concentrations, the HA chains arefar apart, minimizing the hydroxyl and activated carboxylic groups tocome into close contact for reaction to occur. The success of thesemethods to improve gel stability has been only marginal. Moreover, someof these methods do create other problems. For example, the efficiencyof EDC is greatly reduced at high pH therefore crosslinking HA at highpH greatly reduces crosslinking structural robustness of the gel, andwhile crosslinking at very low HA concentration might reduce ester bondformation, it increases inefficient crosslinking of diamines, resultingin gels with too many pendent amine groups.

In accordance with one embodiment of the present invention, formulationmethods are provided for the preparation of stable HA based hydrogelsvia EDC chemistry. Instead of using linear small molecules end cappedwith amino groups, a “4 Arm PEG Amine”, 3-[3-(3-aminopropoxy)-2,2-bis(3-amino-propoxymethyl)-propoxy]-propylamine or 4 AA(see below) is used as the crosslinker.

Unlike diamines with only two amine functionality, 4 AA has four aminefunctional groups which can all be used in crosslinking. Unlike withdiamines where the best case scenario is for a molecule of diamine tocrosslink a maximum of two HA chains, one molecule of 4 AA can crosslinka maximum of 4 HA chains. The use of 4 AA has been found to greatlyenhance crosslinking efficiency, resulting in robust hydrogels that areeasily sterilized by moist steam without fear of imparting structuraldamage to the gel.

Also provided herein are methods for preparing HA hydrogels crosslinkedwith HMDA and methods of preparing HA-lysine hydrogels using lysinemethyl ester as the crosslinker. These gels are biocompatible and caneasily be extruded through a syringe and needle for minimal invasiveimplantation.

In one aspect of the invention, dermal filler compositions are providedfor decreasing the appearance of fine lines or superficial wrinkles inthe face.

The compositions generally comprising a biocompatible polymer, forexample, a polysaccharide such as a hyaluronic acid, crosslinked with adi-amine or multiamine crosslinker. The composition is substantiallyoptically transparent and exhibits reduced or no perceptible bluediscoloration when administered into a dermal region of a patient.

The polymer may be selected from the group of polymers consisting ofproteins, peptides and polypeptides, polylysine, collagens,pro-collagens, elastins, and laminins.

The polymer may be selected from the group of polymers consisting ofsynthetic polymers with hydroxyl, amine, and carboxyl functional groups:poly(vinyl alcohol), polyethylene glycol, polyvinlyl amine,polyallylamine, deacetylated polyacrylamide, polyacrylic acid, andpolymethacrylic acid. The polymer may be selected from the group ofpolymers consisting of dentric or branched polymers, including dentricpolyols and dentric polyamines. The polymer may be selected from thegroup of polymers consisting of solid surface with hydroxyl, amine, andcarboxyl functional groups.

The polymer may be a polysaccharide, for example, selected from thegroup of polysaccharides including starch and its derivatives; dextranand its derivatives, cellulose and its derivatives; chitin and chitosanand alginate and its derivatives.

In an exemplary embodiment of the invention, the polymer isglycosaminoglycan. The hydrogel composition disclosed herein can furthercomprise two or more different glycosaminoglycan polymers. As usedherein, the term “glycosaminoglycan” is synonymous with “GAG” and“mucopolysaccharide” and refers to long unbranched polysaccharidesconsisting of a repeating disaccharide units. The repeating unitconsists of a hexose (six-carbon sugar) or a hexuronic acid, linked to ahexosamine (six-carbon sugar containing nitrogen) and pharmaceuticallyacceptable salts thereof. Members of the GAG family vary in the type ofhexosamine, hexose or hexuronic acid unit they contain, such as, e.g.,glucuronic acid, iduronic acid, galactose, galactosamine, glucosamine)and may also vary in the geometry of the glycosidic linkage. Anyglycosaminoglycan polymer is useful in the hydrogel compositionsdisclosed herein with the proviso that the glycosaminoglycan polymerimproves a condition of the skin. Non-limiting examples ofglycosaminoglycans include chondroitin sulfate, dermatan sulfate,keratan sulfate, hyaluronan. Non-limiting examples of an acceptable saltof a glycosaminoglycans includes sodium salts, potassium salts,magnesium salts, calcium salts, and combinations thereof.Glycosaminoglycan and their resulting polymers useful in the hydrogelcompositions and methods disclosed herein are described in, e.g., Pironand Tholin, Polysaccharide Crosslinking, Hydrogel Preparation, ResultingPolysaccharides(s) and Hydrogel(s), uses Thereof, U.S. PatentPublication 2003/0148995; Lebreton, Cross-Linking of Low and HighMolecular Weight Polysaccharides Preparation of Injectable MonophaseHydrogels; Lebreton, Viscoelastic Solutions Containing SodiumHyaluronate and Hydroxypropyl Methyl Cellulose, Preparation and Uses,U.S. Patent Publication 2008/0089918; Lebreton, Hyaluronic Acid-BasedGels Including Lidocaine, U.S. Patent Publication 2010/0028438; andPolysaccharides and Hydrogels thus Obtained, U.S. Patent Publication2006/0194758; and Di Napoli, Composition and Method for Intradermal SoftTissue Augmentation, International Patent Publication WO 2004/073759,each of which is hereby incorporated by reference in its entirety. GAGsuseful in the hydrogel compositions and methods disclosed herein arecommercially available, such as, e.g., hyaluronan-based dermal fillersJUVEDERM®, JUVEDERM® 30, JUVEDERM® Ultra, JUVEDERM® Ultra Plus,JUVEDERM® Ultra XC, and JUVEDERM® Ultra Plus XC (Allergan Inc, Irvine,Calif.). Table 1 lists representative GAGs.

TABLE 1 Examples of GAGs Glycosidic Hexuronic linkage Name acid/HexoseHexosamine geometry Unique features Chondroitin GlcUA or GalNAc or-4GlcUAβ1- Most prevalent GAG sulfate GlcUA(2S) GalNAc(4S) or 3GalNAcβ1-GalNAc(6S) or GalNAc(4S,6S) Dermatan GlcUA or GalNAc or -4IdoUAβ1-Distinguished from sulfate IdoUA or GalNAc(4S) or 3GalNAcβ1- chondroitinsulfate by the IdoUA(2S) GalNAc(6S) or presence of iduronic acid,GalNAc(4S,6S) although some hexuronic acid monosaccharides may beglucuronic acid. Keratan Gal or Gal(6S) GlcNAc or -3Gal(6S)β1- Keratansulfate type II may sulfate GlcNAc(6S) 4GlcNAc(6S)β1- be fucosylated.Heparin GlcUA or GlcNAc or GlcNS or -4IdoUA(2S)α1- Highest negativecharge IdoUA(2S) GlcNAc(6S) or 4GlcNS(6S)α1- density of any knownGlcNS(6S) biological molecule Heparan GlcUA or GlcNAc or GlcNS or-4GlcUAβ1- Highly similar in structure to sulfate IdoUA or GlcNAc(6S) or4GlcNAcα1- heparin, however heparan IdoUA(2S) GlcNS(6S) sulfatesdisaccharide units are organized into distinct sulfated and non-sulfateddomains. Hyaluronan GlcUA GlcNAc -4GlcUAβ1- The only GAG that is3GlcNAcβ1- exclusively non-sulfated GlcUA = β-D-glucuronic acidGlcUA(2S) = 2-O-sulfo-β-D-glucuronic acid IdoUA = α-L-iduronic acidIdoUA(2S) = 2-O-sulfo-α-L-iduronic acid Gal = β-D-galactose Gal(6S) =6-O-sulfo-β-D-galactose GalNAc = β-D-N-acetylgalactosamine GalNAc(4S) =β-D-N-acetylgalactosamine-4-O-sulfate GalNAc(6S) =β-D-N-acetylgalactosamine-6-O-sulfate GalNAc(4S,6S) =β-D-N-acetylgalactosamine-4-O, 6-O-sulfate GlcNAc =α-D-N-acetylglucosamine GlcNS = α-D-N-sulfoglucosamine GlcNS(6S) =α-D-N-sulfoglucosamine-6-O-sulfate

Aspects of the present specification provide, in part, a hydrogelcomposition comprising a hyaluronan polymer. As used herein, the term“hyaluronic acid polymer” is synonymous with “HA polymer”, “hyaluronicacid polymer”, and “hyaluronate polymer” refers to an anionic,non-sulfated glycosaminoglycan polymer comprising disaccharide units,which themselves include D-glucuronic acid and D-N-acetylglucosaminemonomers, linked together via alternating β-1,4 and β-1,3 glycosidicbonds and pharmaceutically acceptable salts thereof. Hyaluronan polymerscan be purified from animal and non-animal sources. Polymers ofhyaluronan can range in size from about 5,000 Da to about 20,000,000 Da.Any hyaluronan polymer is useful in the compositions disclosed hereinwith the proviso that the hyaluronan improves a condition of the skin.Non-limiting examples of pharmaceutically acceptable salts of hyaluronaninclude sodium hyaluronan, potassium hyaluronan, magnesium hyaluronan,calcium hyaluronan, and combinations thereof.

Aspects of the present specification provide, in part, a hydrogelcomposition comprising a crosslinked glycosaminoglycan polymer. As usedherein, the term “crosslinked” refers to the intermolecular bondsjoining the individual polymer molecules, or monomer chains, into a morestable structure like a gel. As such, a crosslinked glycosaminoglycanpolymer has at least one intermolecular bond joining at least oneindividual polymer molecule to another one. The crosslinking ofglycosaminoglycan polymers typically result in the formation of ahydrogel. Such hydrogels have high viscosity and require considerableforce to extrude through a fine needle.

Glycosaminoglycan polymers disclosed herein may be crosslinked usingdialdehydes and disulfides crosslinking agents including, withoutlimitation, multifunctional PEG-based crosslinking agents, divinylsulfones, diglycidyl ethers, and bis-epoxides, biscarbodiimide.Non-limiting examples of hyaluronan crosslinking agents includemultifunctional PEG-based crosslinking agents like pentaerythritoltetraglycidyl ether (PETGE), divinyl sulfone (DVS), 1,4-butanedioldiglycidyl ether (BDDE), 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE),1,2,7,8-diepoxyoctane (DEO), (phenylenebis-(ethyl)-carbodiimide and 1,6hexamethylenebis (ethylcarbodiimide), adipic dihydrazide (ADH),bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (HMDA),1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, lysine, lysine methyl ester,or combinations thereof.

In other aspects of the invention, the crosslinker is a di-amine ormultiamine crosslinker. In another aspect, the crosslinker is made up ofat least three and a most eight PEG chains emanating from a centralpoint, each chain having a terminal amine group. The crosslinker maycontain PEG chains having a least one ethylene glycol unit and not morethan 55 ethylene glycol units.

Other useful cross-linking agents are disclosed in Stroumpoulis andTezel, Tunably Crosslinked Polysaccharide Compositions, U.S. patentapplication Ser. No. 12/910,466, filed Oct. 22, 2010, which isincorporated by reference in its entirety. Non-limiting examples ofmethods of crosslinking glycosaminoglycan polymers are described in,e.g., Glycosaminoglycan polymers useful in the compositions and methodsdisclosed herein are described in, e.g., Piron and Tholin,Polysaccharide Crosslinking, Hydrogel Preparation, ResultingPolysaccharides(s) and Hydrogel(s), uses Thereof, U.S. PatentPublication 2003/0148995; Lebreton, Cross-Linking of Low and HighMolecular Weight Polysaccharides Preparation of Injectable MonophaseHydrogels; Lebreton, Viscoelastic Solutions Containing SodiumHyaluronate and Hydroxypropyl Methyl Cellulose, Preparation and Uses,U.S. Patent Publication 2008/0089918; Lebreton, Hyaluronic Acid-BasedGels Including Lidocaine, U.S. Patent Publication 2010/0028438; andPolysaccharides and Hydrogels thus Obtained, U.S. Patent Publication2006/0194758; and Di Napoli, Composition and Method for Intradermal SoftTissue Augmentation, International Patent Publication WO 2004/073759,each of which is hereby incorporated by reference in its entirety.

Aspects of the present specification provide, in part, a hydrogelcomposition comprising a crosslinked glycosaminoglycan polymer having adegree of crosslinking. As used herein, the term “degree ofcrosslinking” refers to the percentage of glycosaminoglycan polymermonomeric units, such as, e.g., the disaccharide monomer units ofhyaluronan that are bound to a cross-linking agent. The degree ofcrosslinking is expressed as the percent weight ratio of thecrosslinking agent to glycosaminoglycan. The degree of crosslinking incertain advantageous embodiment of the invention is between about 3% andabout 12%, for example, between about 5% and about 10%. The crosslinkeris present in the composition at a concentration between about 1 μM toabout 100 μM, for example, between about 10 μM to about 50 μM.

In an embodiment, a hydrogel composition comprises a crosslinkedglycosaminoglycan polymer, for example, crosslinked hyaluronic acid,wherein the crosslinked glycosaminoglycan polymer is present in thecomposition at a concentration of, for example, between about 10 mg/mland about 40 mg/ml, for example, between about 18 mg/ml and about 30mg/ml. In some embodiments, the compositions have a total hyaluronicacid concentration of about 22 mg/ml, about 23 mg/ml, about 24 mg/ml orabout 25 mg/ml.

Aspects of the present specification provide, in part, a hydrogelcomposition comprising hyaluronan polymers of low molecular weight,hyaluronan polymers of high molecular weight, or hyaluronan polymers ofboth low and high molecular weight.

As used herein, the term “high molecular weight” when referring to“hyaluronan” refers to hyaluronan polymers having a mean molecularweight of 1,000,000 Da or greater. Non-limiting examples of a highmolecular weight hyaluronan polymers include hyaluronan polymers about1,500,000 Da, about 2,000,000 Da, about 2,500,000 Da, about 3,000,000Da, about 3,500,000 Da, about 4,000,000 Da, about 4,500,000 Da, andabout 5,000,000 Da. As used herein, the term “low molecular weight” whenreferring to “hyaluronan” refers to hyaluronan polymers having a meanmolecular weight of less than 1,000,000 Da. Non-limiting examples of alow molecular weight hyaluronan polymers include hyaluronan polymers ofabout 200,000 Da, about 300,000 Da, about 400,000 Da, about 500,000 Da,about 600,000 Da, about 700,000 Da, of about 800,000 Da, and about900,000 Da.

In an embodiment, a composition comprises crosslinked hyaluronanpolymers of low molecular weight. In aspects of this embodiment, acomposition comprises crosslinked hyaluronan polymers having a meanmolecular weight of, e.g., about 100,000 Da, about 200,000 Da, about300,000 Da, about 400,000 Da, about 500,000 Da, about 600,000 Da, about700,000 Da, about 800,000 Da, or about 900,000 Da. In yet other aspectsof this embodiment, a composition comprises crosslinked hyaluronanpolymers having a mean molecular weight of, e.g., at most 100,000 Da, atmost 200,000 Da, at most 300,000 Da, at most 400,000 Da, at most 500,000Da, at most 600,000 Da, at most 700,000 Da, at most 800,000 Da, at most900,000 Da, or at most 950,000 Da. In still other aspects of thisembodiment, a composition comprises crosslinked hyaluronan polymershaving a mean molecular weight of, e.g., about 100,000 Da to about500,000 Da, about 200,000 Da to about 500,000 Da, about 300,000 Da toabout 500,000 Da, about 400,000 Da to about 500,000 Da, about 500,000 Dato about 950,000 Da, about 600,000 Da to about 950,000 Da, about 700,000Da to about 950,000 Da, about 800,000 Da to about 950,000 Da, about300,000 Da to about 600,000 Da, about 300,000 Da to about 700,000 Da,about 300,000 Da to about 800,000 Da, or about 400,000 Da to about700,000 Da.

In another embodiment, a composition comprises crosslinked hyaluronanpolymers of high molecular weight. In aspects of this embodiment, acomposition comprises a crosslinked hyaluronan polymers having a meanmolecular weight of, e.g., about 1,000,000 Da, about 1,500,000 Da, about2,000,000 Da, about 2,500,000 Da, about 3,000,000 Da, about 3,500,000Da, about 4,000,000 Da, about 4,500,000 Da, or about 5,000,000 Da. Inyet other aspects of this embodiment, a composition comprises acrosslinked hyaluronan polymers having a mean molecular weight of, e.g.,at least 1,000,000 Da, at least 1,500,000 Da, at least 2,000,000 Da, atleast 2,500,000 Da, at least 3,000,000 Da, at least 3,500,000 Da, atleast 4,000,000 Da, at least 4,500,000 Da, or at least 5,000,000 Da. Instill other aspects of this embodiment, a composition comprises acrosslinked hyaluronan polymers having a mean molecular weight of, e.g.,about 1,000,000 Da to about 5,000,000 Da, about 1,500,000 Da to about5,000,000 Da, about 2,000,000 Da to about 5,000,000 Da, about 2,500,000Da to about 5,000,000 Da, about 2,000,000 Da to about 3,000,000 Da,about 2,500,000 Da to about 3,000,000 Da.

In yet another embodiment, a composition comprises a crosslinkedhyaluronan polymers where the crosslinked hyaluronan polymers comprise acombination of both high molecular weight hyaluronan polymers and lowmolecular weight hyaluronan polymers, in various ratios. In aspects ofthis embodiment, a composition comprises a crosslinked hyaluronanpolymers where the crosslinked hyaluronan polymers comprises acombination of both high molecular weight hyaluronan polymers and lowmolecular weight hyaluronan polymers in a ratio of about 20:1, about15:1, about 10:1, about 5:1, about 1:1, about 1:5 about 1:10, about1:15, or about 1:20.

Aspects of the present specification provide, in part, a hydrogelcomposition comprising an uncrosslinked glycosaminoglycan polymer. Asused herein, the term “uncrosslinked” refers to a lack of intermolecularbonds joining the individual glycosaminoglycan polymer molecules, ormonomer chains. As such, an uncrosslinked glycosaminoglycan polymer isnot linked to any other glycosaminoglycan polymer by an intermolecularbond. In aspects of this embodiment, a composition comprises anuncrosslinked chondroitin sulfate polymer, an uncrosslinked dermatansulfate polymer, an uncrosslinked keratan sulfate polymer, anuncrosslinked heparan polymer, an uncrosslinked heparan sulfate polymer,or an uncrosslinked hyaluronan polymer. Uncrosslinked glycosaminoglycanpolymers are water soluble and generally remain fluid in nature. Assuch, uncross-linked glycosaminoglycan polymers are often mixed with aglycosaminoglycan polymer-based hydrogel composition as a lubricant tofacilitate the extrusion process of the composition through a fineneedle.

In an embodiment, a composition comprises an uncrosslinkedglycosaminoglycan polymer where the uncrosslinked glycosaminoglycanpolymer is present at a concentration of, e.g., about 2 mg/g, about 3mg/g, about 4 mg/g, about 5 mg/g, about 6 mg/g, about 7 mg/g, about 8mg/g, about 9 mg/g, about 10 mg/g, about 11 mg/g, about 12 mg/g, about13 mg/g, about 13.5 mg/g, about 14 mg/g, about 15 mg/g, about 16 mg/g,about 17 mg/g, about 18 mg/g, about 19 mg/g, about 20 mg/g, about 40mg/g, or about 60 mg/g. In other aspects of this embodiment, acomposition comprises an uncrosslinked glycosaminoglycan where theuncrosslinked glycosaminoglycan is present at a concentration of, e.g.,at least 1 mg/g, at least 2 mg/g, at least 3 mg/g, at least 4 mg/g, atleast 5 mg/g, at least 10 mg/g, at least 15 mg/g, at least 20 mg/g, atleast 25 mg/g at least 35 mg/g, or at least 40 mg/g. In yet otheraspects of this embodiment, a composition comprises an uncrosslinkedglycosaminoglycan where the uncrosslinked glycosaminoglycan is presentat a concentration of, e.g., at most 1 mg/g, at most 2 mg/g, at most 3mg/g, at most 4 mg/g, at most 5 mg/g, at most 10 mg/g, at most 15 mg/g,at most 20 mg/g, or at most 25 mg/g. In still other aspects of thisembodiment, a composition comprises an uncrosslinked glycosaminoglycanwhere the uncrosslinked glycosaminoglycan is present at a concentrationof, e.g., about 1 mg/g to about 60 mg/g, about 10 mg/g to about 40 mg/g,about 7.5 mg/g to about 19.5 mg/g, about 8.5 mg/g to about 18.5 mg/g,about 9.5 mg/g to about 17.5 mg/g, about 10.5 mg/g to about 16.5 mg/g,about 11.5 mg/g to about 15.5 mg/g, or about 12.5 mg/g to about 14.5mg/g.

Aspects of the present specification provide, in part, a hydrogelcomposition comprising a ratio of crosslinked glycosaminoglycan polymerand uncrosslinked glycosaminoglycan polymer. This ratio of crosslinkedand uncrosslinked glycosaminoglycan polymer is also known as thegel:fluid ratio. Any gel:fluid ratio is useful in making thecompositions disclosed herein with the proviso that such ratio producesa composition disclosed herein that improves a skin condition asdisclosed herein. Non-limiting examples of gel:fluid ratios incompositions of the present invention include 100:0, 98:2, 90:10, 75:25,70:30, 60:40, 50:50, 40:60, 30:70, 25:75, 10:90; 2:98, and 0:100.

In aspects of this embodiment, a composition comprises a crosslinkedglycosaminoglycan polymer and an uncrosslinked glycosaminoglycan polymerwhere the gel:fluid ratio is, e.g., about 0:100, about 1:99, about 2:98,about 3:97, about 4:96, about 5:95, about 6:94, about 7:93, about 8:92,about 9:91, or about 10:90. In other aspects of this embodiment, acomposition comprises a crosslinked glycosaminoglycan polymer and anuncrosslinked glycosaminoglycan polymer where the gel:fluid ratio is,e.g., at most 1:99, at most 2:98, at most 3:97, at most 4:96, at most5:95, at most 6:94, at most 7:93, at most 8:92, at most 9:91, or at most10:90. In yet other aspects of this embodiment, a composition comprisesa crosslinked glycosaminoglycan polymer and an uncrosslinkedglycosaminoglycan polymer where the gel:fluid ratio is, e.g., about0:100 to about 3:97, about 0:100 to about 5:95, or about 0:100 to about10:90.

A hydrogel composition disclosed herein may further comprise anotheragent or combination of agents that provide a beneficial effect when thecomposition is administered to an individual. Such beneficial agentsinclude, without limitation, an antioxidant, an anti-itch agent, ananti-cellulite agent, an anti-scarring agent, an anti-inflammatoryagent, an anesthetic agent, an anti-irritant agent, a vasoconstrictor, avasodilator, an anti-hemorrhagic agent like a hemostatic agent oranti-fibrinolytic agent, a desquamating agent, a tensioning agent, ananti-acne agent, a pigmentation agent, an anti-pigmentation agent, or amoisturizing agent.

For purposes of the present specification, unless otherwise stated, “%”in a formulation is defined as weight by weight (i.e., w/w) percentage.

Aspects of the present specification provide, in part, a hydrogelcomposition disclosed herein that may comprise a coupling agent. Thecoupling agent may be a water soluble coupling agent, for example, awater soluble carbodiimide. In one embodiment, the coupling agent is1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC). Theconcentration of the coupling agent may be between about 10 μM to about50 μM

Aspects of the present specification provide, in part, that a couplingagent may be used in conjunction with a water soluble activating agent.The activating agent may be N-hydroxysuccinimide (NHS) orN-hydroxysulfosuccinimide (sulfoNHS). The activating agent is present ata concentration between about 5 μM to about 25 μM.

Aspects of the present specification provide, in part, a hydrogelcomposition disclosed herein that may optionally comprise an anestheticagent. An anesthetic agent is preferably a local anesthetic agent, i.e.,an anesthetic agent that causes a reversible local anesthesia and a lossof nociception, such as, e.g., aminoamide local anesthetics andaminoester local anesthetics. The amount of an anesthetic agent includedin a composition disclosed herein is an amount effective to mitigatepain experienced by an individual upon administration of thecomposition. As such, the amount of an anesthetic agent included in acomposition disclosed in the present specification is between about 0.1%to about 5% by weight of the total composition. Non-limiting examples ofanesthetic agents include lidocaine, ambucaine, amolanone, amylocalne,benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine,butacaine, butamben, butanilicaine, butethamine, butoxycaine,carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine,dibucaine, dimethysoquin, dimethocaine, diperodon, dycyclonine,ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine,euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine,isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine,mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine,naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine,phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine,prilocalne, procaine, propanocaine, proparacaine, propipocaine,propoxycaine, psuedococaine, pyrrocaine, ropivacaine, salicyl alcohol,tetracaine, tolycaine, trimecaine, zolamine, combinations thereof, andsalts thereof. Non-limiting examples of aminoester local anestheticsinclude procaine, chloroprocaine, cocaine, cyclomethycaine, cimethocaine(larocaine), propoxycaine, procaine (novocaine), proparacaine,tetracaine (amethocaine). Non-limiting examples of aminoamide localanesthetics include articaine, bupivacaine, cinchocaine (dibucaine),etidocaine, levobupivacaine, lidocaine (lignocaine), mepivacaine,piperocaine, prilocalne, ropivacaine, and trimecaine. A compositiondisclosed herein may comprise a single anesthetic agent or a pluralityof anesthetic agents. A non-limiting example of a combination localanesthetic is lidocaine/prilocalne (EMLA).

Thus in an embodiment, a composition disclosed herein comprises ananesthetic agent and salts thereof. In aspects of this embodiment, acomposition disclosed herein comprises an aminoamide local anestheticand salts thereof or an aminoester local anesthetic and salts thereof.In other aspects of this embodiment, a composition disclosed hereincomprises procaine, chloroprocaine, cocaine, cyclomethycaine,cimethocaine, propoxycaine, procaine, proparacaine, tetracaine, or saltsthereof, or any combination thereof. In yet other aspects of thisembodiment, a composition disclosed herein comprises articaine,bupivacaine, cinchocaine, etidocaine, levobupivacaine, lidocaine,mepivacaine, piperocaine, prilocalne, ropivacaine, trimecaine, or saltsthereof, or any combination thereof. In still other aspects of thisembodiment, a composition disclosed herein comprises alidocaine/prilocalne combination.

In other aspects of this embodiment, a composition disclosed hereincomprises an anesthetic agent in an amount of, e.g., about 0.1%, about0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about0.8% about 0.9%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10% byweight of the total composition. In yet other aspects, a compositiondisclosed herein comprises an anesthetic agent in an amount of, e.g., atleast 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%,at least 0.6%, at least 0.7%, at least 0.8% at least 0.9%, at least1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, atleast 6.0%, at least 7.0%, at least 8.0%, at least 9.0%, or at least 10%by weight of the total composition. In still other aspects, acomposition disclosed herein comprises an anesthetic agent in an amountof, e.g., at most 0.1%, at most 0.2%, at most 0.3%, at most 0.4%, atmost 0.5%, at most 0.6%, at most 0.7%, at most 0.8% at most 0.9%, atmost 1.0%, at most 2.0%, at most 3.0%, at most 4.0%, at most 5.0%, atmost 6.0%, at most 7.0%, at most 8.0%, at most 9.0%, or at most 10% byweight of the total composition. In further aspects, a compositiondisclosed herein comprises an anesthetic agent in an amount of, e.g.,about 0.1% to about 0.5%, about 0.1% to about 1.0%, about 0.1% to about2.0%, about 0.1% to about 3.0%, about 0.1% to about 4.0%, about 0.1% toabout 5.0%, about 0.2% to about 0.9%, about 0.2% to about 1.0%, about0.2% to about 2.0%, about 0.5% to about 1.0%, or about 0.5% to about2.0% by weight of the total composition.

In one aspect of the present invention, an injectable dermal filler isprovided which comprises a polymer, for example, a glycosaminoglycanpolymer, for example a hylaluronic acid polymer, for example, ahyaluronic acid at least a portion of which is crosslinked, and anadditive or beneficial agent combined with the polymer.

Aspects of the present specification provide, in part, a hydrogelcomposition disclosed herein that exhibits an elastic modulus or storagemodulus. The compositions as disclosed herein are viscoelastic in thatthe composition has an elastic component (solid-like such as, e.g.,crosslinked glycosaminoglycan polymers) and a viscous component(liquid-like such as, e.g., uncrosslinked glycosaminoglycan polymers ora carrier phase) when a force is applied (stress, deformation).

Elastic modulus, or modulus of elasticity, refers to the ability of ahydrogel material to resists deformation, or, conversely, an object'stendency to be non-permanently deformed when a force is applied to it.Elastic modulus characterizes the firmness of a composition and is alsoknown as the storage modulus because it describes the storage of energyfrom the motion of the composition. The elastic modulus describes theinteraction between elasticity and strength (G′=stress/strain) and, assuch, provides a quantitative measurement of a composition's hardness orsoftness. The elastic modulus of an object is defined as the slope ofits stress-strain curve in the elastic deformation region:λ=stress/strain, where λ is the elastic modulus in Pascal's; stress isthe force causing the deformation divided by the area to which the forceis applied; and strain is the ratio of the change caused by the stressto the original state of the object. Although depending on the speed atwhich the force is applied, a stiffer composition will have a higherelastic modulus and it will take a greater force to deform the materiala given distance, such as, e.g., an injection. Specifying how stressesare to be measured, including directions, allows for many types ofelastic moduli to be defined. The three primary elastic moduli aretensile modulus, shear modulus, and bulk modulus.

In aspects of this embodiment, a hydrogel composition exhibits anelastic modulus of, e.g., at least about 20 Pa to about 3000 Pa. Forexample the composition exhibits an elastic modulus of between, at leastabout 50 Pa to about 2500 Pa, at least about 100 Pa to about 2000 Pa, atleast about 500 Pa to about 1000 Pa. In other aspects of thisembodiment, a hydrogel composition exhibits an elastic modulus of, e.g.,at least about 20 Pa, at least about 25 Pa, at least about 50 Pa, atleast about 75 Pa, at least about 100 Pa, at least about 125 Pa, atleast about 150 Pa, at least about 175 Pa, at least about 200 Pa, atleast about 250 Pa, at least about 300 Pa, at least about 350 Pa, atleast about 400 Pa, at least about 450 Pa, at least about 500 Pa, atleast about 550 Pa, at least about 600 Pa, at least about 650 Pa, atleast about 700 Pa, at least about 750 Pa, at least about 800 Pa, atleast about 850 Pa, at least about 900 Pa, at least about 950 Pa, atleast about 1,000 Pa, at least about 1,200 Pa, at least about 1,300 Pa,at least about 1,400 Pa, at least about 1,500 Pa, at least 1 about, 600Pa, at least 1 about 700 Pa, at least about 1800 Pa, at least about 1900Pa, at least about 2,000 Pa, at least about 2,100 Pa, at least about2,200 Pa, at least about 2,300 Pa, at least about 2,400 Pa, or at leastabout 2,500 Pa. In yet other aspects of this embodiment, a hydrogelcomposition exhibits an elastic modulus of, e.g., at least about 20 Pato at most about 3000 Pa.

Aspects of the present specification provide, in part, a hydrogelcomposition disclosed herein having a transparency and/or translucency.Optical transparency is the physical property of allowing visible lightto pass through a material, whereas translucency (also calledtranslucence or translucidity) only allows light to pass throughdiffusely. The opposite property is opacity. Transparent materials areclear, while translucent ones cannot be seen through clearly. Thehydrogels disclosed herein may be optically transparent or at leasttranslucent.

In an embodiment, a hydrogel composition disclosed herein is opticallytranslucent. In aspects of this embodiment, a hydrogel compositiondiffusely transmits, e.g., about 75% of the light, about 80% of thelight, about 85% of the light, about 90% of the light, about 95% of thelight, or about 100% of the light. In other aspects of this embodiment,a hydrogel composition diffusely transmits, e.g., at least 75% of thelight, at least 80% of the light, at least 85% of the light, at least90% of the light, or at least 95% of the light. In yet other aspects ofthis embodiment, a hydrogel composition diffusely transmits, e.g., about75% to about 100% of the light, about 80% to about 100% of the light,about 85% to about 100% of the light, about 90% to about 100% of thelight, or about 95% to about 100% of the light. In an embodiment, ahydrogel composition disclosed herein is optically transparent andtransmits 100% of visible light.

A hydrogel composition disclosed herein may be further processed bypulverizing the hydrogel into particles and optionally mixed with acarrier phase such as, e.g., water or a saline solution to form aninjectable or topical substance like a solution, oil, lotion, gel,ointment, cream, slurry, salve, or paste. As such, the disclosedhydrogel compositions may be monophasic or multiphasic compositions. Ahydrogel may be milled to a particle size from about 10 μm to about 1000μm in diameter, such as about 15 μm to about 30 μm, about 50 μm to about75 μm, about 100 μm to about 150 μm, about 200 μm to about 300 μm, about450 μm to about 550 μm, about 600 μm to about 700 μm, about 750 μm toabout 850 μm, or about 900 μm to about 1,000 μm.

Aspects of the present specification provide, in part, a compositiondisclosed herein is injectable. As used herein, the term “injectable”refers to a material having the properties necessary to administer thecomposition into a skin region of an individual using an injectiondevice with a fine needle. As used herein, the term “fine needle” refersto a needle that is 27 gauge or smaller. Injectability of a compositiondisclosed herein can be accomplished by sizing the hydrogel particles asdiscussed above.

In aspect of this embodiment, a hydrogel composition disclosed herein isinjectable through a fine needle. In other aspects of this embodiment, ahydrogel composition disclosed herein is injectable through a needle of,e.g., about 27 gauge, about 30 gauge, or about 32 gauge. In yet otheraspects of this embodiment, a hydrogel composition disclosed herein isinjectable through a needle of, e.g., 22 gauge or smaller, 27 gauge orsmaller, 30 gauge or smaller, or 32 gauge or smaller. In still otheraspects of this embodiment, a hydrogel composition disclosed herein isinjectable through a needle of, e.g., about 22 gauge to about 35 gauge,22 gauge to about 34 gauge, 22 gauge to about 33 gauge, 22 gauge toabout 32 gauge, about 22 gauge to about 27 gauge, or about 27 gauge toabout 32 gauge.

In aspects of this embodiment, a hydrogel composition disclosed hereincan be injected with an extrusion force of about 150 N, about 100 N,about 80 N, about 60 N, about 55 N, about 50 N, about 45 N, about 40 N,about 35 N, about 30 N, about 25 N, about 20 N, or about 15 N at speedsof 100 mm/min. In other aspects of this embodiment, a hydrogelcomposition disclosed herein can be injected through a 27 gauge needlewith an extrusion force of about 150 N or less, about 100 N or less,about 80 N or less, about 60 N or less, about 55 N or less, about 50 Nor less, about 45 N or less, about 40 N or less, about 35 N or less,about 30 N or less, about 25 N or less, about 20 N or less, about 15 Nor less, about 10 N or less, or about 5 N or less. In yet other aspectsof this embodiment, a hydrogel composition disclosed herein can beinjected through a 30 gauge needle with an extrusion force of about 150N or less, about 100 N or less, about 80 N or less, about 60 N or less,about 55 N or less, about 50 N or less, about 45 N or less, about 40 Nor less, about 35 N or less, about 30 N or less, about 25 N or less,about 20 N or less, about 15 N or less, about 10 N or less, or about 5 Nor less. In still other aspects of this embodiment, a hydrogelcomposition disclosed herein can be injected through a 32 gauge needlewith an extrusion force of about 150 N or less, about 100 N or less,about 80 N or less, about 60 N or less, about 55 N or less, about 50 Nor less, about 45 N or less, about 40 N or less, about 35 N or less,about 30 N or less, about 25 N or less, about 20 N or less, about 15 Nor less, about 10 N or less, or about 5 N or less.

Aspects of the present specification provide, in part, a hydrogelcomposition disclosed herein that exhibits cohesivity. Cohesivity, alsoreferred to as cohesion cohesive attraction, cohesive force, orcompression force is a physical property of a material, caused by theintermolecular attraction between like-molecules within the materialthat acts to unite the molecules. Cohesivity is expressed in terms ofgrams-force (gmf). Cohesiveness is affected by, among other factors, themolecular weight ratio of the initial free glycosaminoglycan polymer,the degree of crosslinking of glycosaminoglycan polymers, the amount ofresidual free glycosaminoglycan polymers following crosslinking, and thepH of the hydrogel composition. A composition should be sufficientlycohesive as to remain localized to a site of administration.Additionally, in certain applications, a sufficient cohesiveness isimportant for a composition to retain its shape, and thus functionality,in the event of mechanical load cycling. As such, in one embodiment, ahydrogel composition disclosed herein exhibits cohesivity, on par withwater. In yet another embodiment, a hydrogel composition disclosedherein exhibits sufficient cohesivity to remain localized to a site ofadministration. In still another embodiment, a hydrogel compositiondisclosed herein exhibits sufficient cohesivity to retain its shape. Ina further embodiment, a hydrogel composition disclosed herein exhibitssufficient cohesivity to retain its shape and functionality.

Aspects of the present specification provide, in part, a hydrogelcomposition disclosed herein that exhibits substantial stability. Asused herein, the term “stability” or “stable” when referring to ahydrogel composition disclosed herein refers to a composition that isnot prone to degrading, decomposing, or breaking down to any substantialor significant degree while stored before administration to anindividual. As used herein, the term “substantial heat stability”,“substantially heat stable”, “autoclave stable”, or “steam sterilizationstable” refers to a hydrogel composition disclosed herein that issubstantially stable when subjected to a heat treatment as disclosedherein.

Stability of a hydrogel composition disclosed herein can be determinedby subjecting a hydrogel composition to a heat treatment, such as, e.g.,steam sterilization at normal pressure or under pressure (e.g.,autoclaving). The heat treatment may be carried out at a temperature ofat least about 100° C. for between about one minute and about 10minutes. Substantial stability of a hydrogel composition disclosedherein can be evaluated 1) by determining the change in the extrusionforce (ΔF) of a hydrogel composition disclosed herein aftersterilization, where the change in extrusion force less 2N is indicativeof a substantially stable hydrogel composition as measured by (theextrusion force of a hydrogel composition with the specified additives)minus (the extrusion force of the a hydrogel composition without theadded additives); and/or 2) by determining the change in rheologicalproperties of a hydrogel composition disclosed herein aftersterilization, where the change in tan δ 1 Hz of less than 0.1 isindicative of a substantially stable hydrogel composition as measured by(tan δ 1 Hz of gel formulation with additives) minus (tan δ 1 Hz of gelformulation without additives). As such, a substantially stable hydrogelcomposition disclosed herein retains one or more of the followingcharacteristics after sterilization: homogeneousness, extrusion force,cohesiveness, hyaluronan concentration, agent(s) concentration,osmolarity, pH, or other rheological characteristics desired by thehydrogel before the heat treatment. In one embodiment, the compositionis substantially stable after being sterilized with moist steam attemperatures between about 121° C. to about 124° C. or higher, for about3 min. to about 15 min or more.

In an embodiment, a hydrogel composition comprising a glycosaminoglycanpolymer is processed using a heat treatment that maintains the desiredhydrogel properties disclosed herein. In aspects of this embodiment, ahydrogel composition comprising a glycosaminoglycan polymer and the atleast one agent disclosed herein is processed using a heat treatment of,e.g., about 100° C., about 105° C., about 110° C., about 115° C., about120° C., about 125° C., or about 130° C. In other aspects of thisembodiment, a hydrogel composition comprising a glycosaminoglycanpolymer and the at least one agent disclosed herein is processed using aheat treatment of, e.g., at least 100° C., at least 105° C., at least110° C., at least 115° C., at least 120° C., at least 125° C., or atleast 130° C. In yet other aspects of this embodiment, a hydrogelcomposition comprising a glycosaminoglycan polymer and the at least oneagent disclosed herein is processed using a heat treatment of, e.g.,about 100° C. to about 120° C., about 100° C. to about 125° C., about100° C. to about 130° C., about 100° C. to about 135° C., about 110° C.to about 120° C., about 110° C. to about 125° C., about 110° C. to about130° C., about 110° C. to about 135° C., about 120° C. to about 125° C.,about 120° C. to about 130° C., about 120° C. to about 135° C., about125° C. to about 130° C., or about 125° C. to about 135° C.

Long term stability of a hydrogel composition disclosed herein can bedetermined by subjecting a hydrogel composition to a heat treatment,such as, e.g., storage in an about 45° C. environment for about 60 days.Long term stability of a hydrogel composition disclosed herein can beevaluated 1) by assessing the clarity and color of a hydrogelcomposition after the 45° C. heat treatment, with a clear and uncoloredhydrogel composition being indicative of a substantially stable hydrogelcomposition; 2) by determining the change in the extrusion force (ΔF) ofa hydrogel composition disclosed herein after the 45° C. heat treatment,where the change in extrusion force less 2N is indicative of asubstantially stable hydrogel composition as measured by (the extrusionforce of a hydrogel composition with the specified additives before the45° C. heat treatment) minus (the extrusion force of the a hydrogelcomposition with the specified additives after the 45° C. heattreatment); and/or 3) by determining the change in rheologicalproperties of a hydrogel composition disclosed herein aftersterilization, where the change in tan δ 1 Hz of less than 0.1 isindicative of a substantially stable hydrogel composition as measured by(tan δ 1 Hz of gel formulation with the specified additives before the45° C. heat treatment) minus (tan δ 1 Hz of gel formulation with thespecified additives after the 45° C. heat treatment). As such, a longterm stability of a hydrogel composition disclosed herein is evaluatedby retention of one or more of the following characteristics after the45° C. heat treatment: clarity (transparency and translucency),homogeneousness, and cohesiveness.

In aspects of this embodiment, a hydrogel composition is substantiallystable at room temperature for, e.g., about 3 months, about 6 months,about 9 months, about 12 months, about 15 months, about 18 months, about21 months, about 24 months, about 27 months, about 30 months, about 33months, or about 36 months. In other aspects of this embodiment, ahydrogel composition is substantially stable at room temperature for,e.g., at least 3 months, at least 6 months, at least 9 months, at least12 months, at least 15 months, at least 18 months, at least 21 months,at least 24 months, at least 27 months, at least 30 months, at least 33months, or at least 36 months. In other aspects of this embodiment, ahydrogel composition is substantially stable at room temperature for,e.g., about 3 months to about 12 months, about 3 months to about 18months, about 3 months to about 24 months, about 3 months to about 30months, about 3 months to about 36 months, about 6 months to about 12months, about 6 months to about 18 months, about 6 months to about 24months, about 6 months to about 30 months, about 6 months to about 36months, about 9 months to about 12 months, about 9 months to about 18months, about 9 months to about 24 months, about 9 months to about 30months, about 9 months to about 36 months, about 12 months to about 18months, about 12 months to about 24 months, about 12 months to about 30months, about 12 months to about 36 months, about 18 months to about 24months, about 18 months to about 30 months, or about 18 months to about36 months.

The present compositions may optionally include, without limitation,other pharmaceutically acceptable components, including, withoutlimitation, buffers, preservatives, tonicity adjusters, salts,antioxidants, osmolality adjusting agents, emulsifying agents, wettingagents, and the like.

A pharmaceutically acceptable buffer is a buffer that can be used toprepare a hydrogel composition disclosed herein, provided that theresulting preparation is pharmaceutically acceptable. Non-limitingexamples of pharmaceutically acceptable buffers include acetate buffers,borate buffers, citrate buffers, neutral buffered salines, phosphatebuffers, and phosphate buffered salines. Any concentration of apharmaceutically acceptable buffer can be useful in formulating apharmaceutical composition disclosed herein, with the proviso that atherapeutically effective amount of the active ingredient is recoveredusing this effective concentration of buffer. Non-limiting examples ofconcentrations of physiologically-acceptable buffers occur within therange of about 0.1 mM to about 900 mM. The pH of pharmaceuticallyacceptable buffers may be adjusted, provided that the resultingpreparation is pharmaceutically acceptable. It is understood that acidsor bases can be used to adjust the pH of a pharmaceutical composition asneeded. Any buffered pH level can be useful in formulating apharmaceutical composition, with the proviso that a therapeuticallyeffective amount of the matrix polymer active ingredient is recoveredusing this effective pH level. Non-limiting examples ofphysiologically-acceptable pH occur within the range of about pH 5.0 toabout pH 8.5. For example, the pH of a hydrogel composition disclosedherein can be about 5.0 to about 8.0, or about 6.5 to about 7.5, about7.0 to about 7.4, or about 7.1 to about 7.3.

Pharmaceutically acceptable preservatives include, without limitation,sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole and butylated hydroxytoluene. Pharmaceutically acceptablepreservatives include, without limitation, benzalkonium chloride,chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuricnitrate, a stabilized oxy chloro composition, such as, e.g., PURITE®(Allergan, Inc. Irvine, Calif.) and chelants, such as, e.g., DTPA orDTPA-bisamide, calcium DTPA, and CaNaDTPA-bisamide.

Pharmaceutically acceptable tonicity adjustors useful in a hydrogelcomposition disclosed herein include, without limitation, salts such as,e.g., sodium chloride and potassium chloride; and glycerin. Thecomposition may be provided as a salt and can be formed with many acids,including but not limited to, hydrochloric, sulfuric, acetic, lactic,tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueousor other protonic solvents than are the corresponding free base forms.It is understood that these and other substances known in the art ofpharmacology can be included in a pharmaceutical composition disclosedherein. Other non-limiting examples of pharmacologically acceptablecomponents can be found in, e.g., Ansel, supra, (1999); Gennaro, supra,(2000); Hardman, supra, (2001); and Rowe, supra, (2003), each of whichis hereby incorporated by reference in its entirety.

Aspects of the present specification provide, in part, a method oftreating a soft tissue condition of an individual by administering ahydrogel composition disclosed herein. As used herein, the term“treating,” refers to reducing or eliminating in an individual acosmetic or clinical symptom of a soft tissue condition characterized bya soft tissue imperfection, defect, disease, and/or disorder; ordelaying or preventing in an individual the onset of a cosmetic orclinical symptom of a condition characterized by a soft tissueimperfection, defect, disease, and/or disorder. For example, the term“treating” can mean reducing a symptom of a condition characterized by asoft tissue defect, disease, and/or disorder by, e.g., at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% or at least 100%. The effectiveness of ahydrogel composition disclosed herein in treating a conditioncharacterized by a soft tissue defect, disease, and/or disorder can bedetermined by observing one or more cosmetic, clinical symptoms, and/orphysiological indicators associated with the condition. An improvementin a soft tissue defect, disease, and/or disorder also can be indicatedby a reduced need for a concurrent therapy. Those of skill in the artwill know the appropriate symptoms or indicators associated withspecific soft tissue defect, disease, and/or disorder and will know howto determine if an individual is a candidate for treatment with acompound or composition disclosed herein.

A hydrogel composition in accordance with the invention is administeredto an individual. An individual is typically a human being of any age,gender or race. Typically, any individual who is a candidate for aconventional procedure to treat a soft tissue condition is a candidatefor a method disclosed herein. Although a subject experiencing the signsof aging skin is an adult, subjects experiencing premature aging orother skin conditions suitable for treatment (for example, a scar) canalso be treated with a hydrogel composition disclosed herein. Inaddition, the presently disclosed hydrogel compositions and methods mayapply to individuals seeking a small/moderate enlargement, shape changeor contour alteration of a body part or region, which may not betechnically possible or aesthetically acceptable with existing softtissue implant technology. Pre-operative evaluation typically includesroutine history and physical examination in addition to thoroughinformed consent disclosing all relevant risks and benefits of theprocedure.

The hydrogel composition and methods disclosed herein are useful intreating a soft tissue condition. A soft tissue condition includes,without limitation, a soft tissue imperfection, defect, disease, and/ordisorder. Non-limiting examples of a soft tissue condition includebreast imperfection, defect, disease and/or disorder, such as, e.g., abreast augmentation, a breast reconstruction, mastopexy, micromastia,thoracic hypoplasia, Poland's syndrome, defects due to implantcomplications like capsular contraction and/or rupture; a facialimperfection, defect, disease or disorder, such as, e.g., a facialaugmentation, a facial reconstruction, a mesotherapy, Parry-Rombergsyndrome, lupus erythematosus profundus, dermal divots, scars, sunkenchecks, thin lips, nasal imperfections or defects, retro-orbitalimperfections or defects, a facial fold, line and/or wrinkle like aglabellar line, a nasolabial line, a perioral line, and/or a marionetteline, and/or other contour deformities or imperfections of the face; aneck imperfection, defect, disease or disorder; a skin imperfection,defect, disease and/or disorder; other soft tissue imperfections,defects, diseases and/or disorders, such as, e.g., an augmentation or areconstruction of the upper arm, lower arm, hand, shoulder, back, torsoincluding abdomen, buttocks, upper leg, lower leg including calves, footincluding plantar fat pad, eye, genitals, or other body part, region orarea, or a disease or disorder affecting these body parts, regions orareas; urinary incontinence, fecal incontinence, other forms ofincontinence; and gastroesophageal reflux disease (GERD). As usedherein, the term “mesotherapy” refers to a non-surgical cosmetictreatment technique of the skin involving intra-epidermal, intra-dermal,and/or subcutaneous injection of an agent administered as small multipledroplets into the epidermis, dermo-epidermal junction, and/or thedermis.

The amount of a hydrogel composition used with any of the methods asdisclosed herein will typically be determined based on the alterationand/or improvement desired, the reduction and/or elimination of a softtissue condition symptom desired, the clinical and/or cosmetic effectdesired by the individual and/or physician, and the body part or regionbeing treated. The effectiveness of composition administration may bemanifested by one or more of the following clinical and/or cosmeticmeasures: altered and/or improved soft tissue shape, altered and/orimproved soft tissue size, altered and/or improved soft tissue contour,altered and/or improved tissue function, tissue ingrowth support and/ornew collagen deposition, sustained engraftment of composition, improvedpatient satisfaction and/or quality of life, and decreased use ofimplantable foreign material.

Effectiveness of the compositions and methods in treating a facial softtissue may be manifested by one or more of the following clinical and/orcosmetic measures: increased size, shape, and/or contour of facialfeature like increased size, shape, and/or contour of lip, cheek or eyeregion; altered size, shape, and/or contour of facial feature likealtered size, shape, and/or contour of lip, cheek or eye region shape;reduction or elimination of a wrinkle, fold or line in the skin;resistance to a wrinkle, fold or line in the skin; rehydration of theskin; increased elasticity to the skin; reduction or elimination of skinroughness; increased and/or improved skin tautness; reduction orelimination of stretch lines or marks; increased and/or improved skintone, shine, brightness and/or radiance; increased and/or improved skincolor, reduction or elimination of skin paleness; sustained engraftmentof composition; decreased side effects; improved patient satisfactionand/or quality of life.

As yet another example, for urinary incontinence procedures,effectiveness of the compositions and methods for sphincter support maybe manifested by one or more of the following clinical measures:decreased frequency of incontinence, sustained engraftment, improvedpatient satisfaction and/or quality of life, and decreased use ofimplantable foreign filler.

In aspects of this embodiment, the amount of a hydrogel compositionadministered is, e.g., about 0.01 g, about 0.05 g, about 0.1 g, about0.5 g, about 1 g, about 5 g, about 10 g, about 20 g, about 30 g, about40 g, about 50 g, about 60 g, about 70 g, about 80 g, about 90 g, about100 g, about 150 g, or about 200 g. In other aspects of this embodiment,the amount of a hydrogel composition administered is, e.g., about 0.01 gto about 0.1 g, about 0.1 g to about 1 g, about 1 g to about 10 g, about10 g to about 100 g, or about 50 g to about 200 g. In yet other aspectsof this embodiment, the amount of a hydrogel composition administeredis, e.g., about 0.01 mL, about 0.05 mL, about 0.1 mL, about 0.5 mL,about 1 mL, about 5 mL, about 10 mL, about 20 mL, about 30 mL, about 40mL, about 50 mL, about 60 mL, about 70 g, about 80 mL, about 90 mL,about 100 mL, about 150 mL, or about 200 mL. In other aspects of thisembodiment, the amount of a hydrogel composition administered is, e.g.,about 0.01 mL to about 0.1 mL, about 0.1 mL to about 1 mL, about 1 mL toabout 10 mL, about 10 mL to about 100 mL, or about 50 mL to about 200mL.

The duration of treatment will typically be determined based on thecosmetic and/or clinical effect desired by the individual and/orphysician and the body part or region being treated. In aspects of thisembodiment, administration of a hydrogel composition disclosed hereincan treat a soft tissue condition for, e.g., about 6 months, about 7months, about 8 months, about 9 months, about 10 months, about 11months, about 12 months, about 13 months, about 14 months, about 15months, about 18 months, or about 24 months. In other aspects of thisembodiment, administration of a hydrogel composition disclosed hereincan treat a soft tissue condition for, e.g., at least 6 months, at least7 months, at least 8 months, at least 9 months, at least 10 months, atleast 11 months, at least 12 months, at least 13 months, at least 14months, at least 15 months, at least 18 months, or at least 24 months.In yet aspects of this embodiment, administration of a hydrogelcomposition disclosed herein can treat a soft tissue condition for,e.g., about 6 months to about 12 months, about 6 months to about 15months, about 6 months to about 18 months, about 6 months to about 21months, about 6 months to about 24 months, about 9 months to about 12months, about 9 months to about 15 months, about 9 months to about 18months, about 9 months to about 21 months, about 6 months to about 24months, about 12 months to about 15 months, about 12 months to about 18months, about 12 months to about 21 months, about 12 months to about 24months, about 15 months to about 18 months, about 15 months to about 21months, about 15 months to about 24 months, about 18 months to about 21months, about 18 months to about 24 months, or about 21 months to about24 months.

Aspects of the present specification provide, in part, administering ahydrogel composition disclosed herein. As used herein, the term“administering” means any delivery mechanism that provides a compositiondisclosed herein to an individual that potentially results in aclinically, therapeutically, or experimentally beneficial result. Theactual delivery mechanism used to administer a composition to anindividual can be determined by a person of ordinary skill in the art bytaking into account factors, including, without limitation, the type ofskin condition, the location of the skin condition, the cause of theskin condition, the severity of the skin condition, the degree of reliefdesired, the duration of relief desired, the particular compositionused, the rate of excretion of the particular composition used, thepharmacodynamics of the particular composition used, the nature of theother compounds included in the particular composition used, theparticular route of administration, the particular characteristics,history and risk factors of the individual, such as, e.g., age, weight,general health and the like, or any combination thereof. In an aspect ofthis embodiment, a composition disclosed herein is administered to askin region of an individual by injection.

The route of administration of a hydrogel composition to an individualpatient will typically be determined based on the cosmetic and/orclinical effect desired by the individual and/or physician and the bodypart or region being treated. A composition disclosed herein may beadministered by any means known to persons of ordinary skill in the artincluding, without limitation, syringe with needle, a pistol (forexample, a hydropneumatic-compression pistol), catheter, topically, orby direct surgical implantation. The hydrogel composition disclosedherein can be administered into a skin region such as, e.g., a dermalregion or a hypodermal region. For example, a hydrogel compositiondisclosed herein can be injected utilizing needles with a diameter ofabout 0.26 mm to about 0.4 mm and a length ranging from about 4 mm toabout 14 mm. Alternately, the needles can be 21 to 32 G and have alength of about 4 mm to about 70 mm. Preferably, the needle is asingle-use needle. The needle can be combined with a syringe, catheter,and/or a pistol.

In addition, a composition disclosed herein can be administered once, orover a plurality of times. Ultimately, the timing used will followquality care standards. For example, a hydrogel composition disclosedherein can be administered once or over several sessions with thesessions spaced apart by a few days, or weeks. For instance, anindividual can be administered a hydrogel composition disclosed hereinevery 1, 2, 3, 4, 5, 6, or 7 days or every 1, 2, 3, or 4 weeks. Theadministration a hydrogel composition disclosed herein to an individualcan be on a monthly or bi-monthly basis or administered every 3, 6, 9,or 12 months.

Aspects of the present specification provide, in part, a dermal region.As used herein, the term “dermal region” refers to the region of skincomprising the epidermal-dermal junction and the dermis including thesuperficial dermis (papillary region) and the deep dermis (reticularregion). The skin is composed of three primary layers: the epidermis,which provides waterproofing and serves as a barrier to infection; thedermis, which serves as a location for the appendages of skin; and thehypodermis (subcutaneous adipose layer). The epidermis contains no bloodvessels, and is nourished by diffusion from the dermis. The main type ofcells which make up the epidermis are keratinocytes, melanocytes,Langerhans cells and Merkels cells.

The dermis is the layer of skin beneath the epidermis that consists ofconnective tissue and cushions the body from stress and strain. Thedermis is tightly connected to the epidermis by a basement membrane. Italso harbors many Mechanoreceptor/nerve endings that provide the senseof touch and heat. It contains the hair follicles, sweat glands,sebaceous glands, apocrine glands, lymphatic vessels and blood vessels.The blood vessels in the dermis provide nourishment and waste removalfrom its own cells as well as from the Stratum basale of the epidermis.The dermis is structurally divided into two areas: a superficial areaadjacent to the epidermis, called the papillary region, and a deepthicker area known as the reticular region.

The papillary region is composed of loose areolar connective tissue. Itis named for its fingerlike projections called papillae that extendtoward the epidermis. The papillae provide the dermis with a “bumpy”surface that interdigitates with the epidermis, strengthening theconnection between the two layers of skin. The reticular region liesdeep in the papillary region and is usually much thicker. It is composedof dense irregular connective tissue, and receives its name from thedense concentration of collagenous, elastic, and reticular fibers thatweave throughout it. These protein fibers give the dermis its propertiesof strength, extensibility, and elasticity. Also located within thereticular region are the roots of the hair, sebaceous glands, sweatglands, receptors, nails, and blood vessels. Tattoo ink is held in thedermis. Stretch marks from pregnancy are also located in the dermis.

The hypodermis lies below the dermis. Its purpose is to attach thedermal region of the skin to underlying bone and muscle as well assupplying it with blood vessels and nerves. It consists of looseconnective tissue and elastin. The main cell types are fibroblasts,macrophages and adipocytes (the hypodermis contains 50% of body fat).Fat serves as padding and insulation for the body.

In an aspect of this embodiment, a hydrogel composition disclosed hereinis administered to a skin region of an individual by injection into adermal region or a hypodermal region. In aspects of this embodiment, ahydrogel composition disclosed herein is administered to a dermal regionof an individual by injection into, e.g., an epidermal-dermal junctionregion, a papillary region, a reticular region, or any combinationthereof.

Advantageously, some of the present compositions are especially usefuland effective in reducing appearance of fine lines, for example, in thinskin regions, of a patient. The skin region treated may be any skinregion having fine lines or wrinkling, for example, due to age or UVexposure, and can be especially useful for treating, for example, andsmoothing the appearance of, the tear trough region, forehead region,glabellar lines, or periorbital region.

For example, methods are provided for fine line treatment comprising thesteps of administering to a dermal region of a patient a dermal fillercomposition as described elsewhere herein, at a depth of no greater thanabout 1 mm. When so administered, the compositions of these embodimentsexhibit reduced or no perceptible blue discoloration when administeredat a depth of no greater than about 1.0 mm, no greater than about 0.8mm, no greater than about 0.6 mm, or no greater than about 4 mm or lessinto the dermal region.

Other aspects of the present specification disclose, in part, a methodof treating a skin condition comprises the step of administering to anindividual suffering from a skin condition a hydrogel compositiondisclosed herein, wherein the administration of the composition improvesthe skin condition, thereby treating the skin condition. In an aspect ofthis embodiment, a skin condition is a method of treating skindehydration comprises the step of administering to an individualsuffering from skin dehydration a hydrogel composition disclosed herein,wherein the administration of the composition rehydrates the skin,thereby treating skin dehydration. In another aspect of this embodiment,a method of treating a lack of skin elasticity comprises the step ofadministering to an individual suffering from a lack of skin elasticitya hydrogel composition disclosed herein, wherein the administration ofthe composition increases the elasticity of the skin, thereby treating alack of skin elasticity. In yet another aspect of this embodiment, amethod of treating skin roughness comprises the step of administering toan individual suffering from skin roughness a hydrogel compositiondisclosed herein, wherein the administration of the compositiondecreases skin roughness, thereby treating skin roughness. In stillanother aspect of this embodiment, a method of treating a lack of skintautness comprises the step of administering to an individual sufferingfrom a lack of skin tautness a hydrogel composition disclosed herein,wherein the administration of the composition makes the skin tauter,thereby treating a lack of skin tautness.

In a further aspect of this embodiment, a method of treating a skinstretch line or mark comprises the step of administering to anindividual suffering from a skin stretch line or mark a hydrogelcomposition disclosed herein, wherein the administration of thecomposition reduces or eliminates the skin stretch line or mark, therebytreating a skin stretch line or mark. In another aspect of thisembodiment, a method of treating skin paleness comprises the step ofadministering to an individual suffering from skin paleness a hydrogelcomposition disclosed herein, wherein the administration of thecomposition increases skin tone or radiance, thereby treating skinpaleness. In another aspect of this embodiment, a method of treatingskin wrinkles comprises the step of administering to an individualsuffering from skin wrinkles a hydrogel composition disclosed herein,wherein the administration of the composition reduces or eliminates skinwrinkles, thereby treating skin wrinkles. In yet another aspect of thisembodiment, a method of treating skin wrinkles comprises the step ofadministering to an individual a hydrogel composition disclosed herein,wherein the administration of the composition makes the skin resistantto skin wrinkles, thereby treating skin wrinkles.

In one aspect of the invention, dermal fillers are provided which areespecially effective in treating and eliminating the appearance of finelines, for example, relatively superficial, creases in the skin, forexample, but not limited to, fine lines near the eyes, the tear troughregion, forehead, periobital, glabellar lines, etc.

The appearance of a blue discoloration at the skin site where a dermalfiller had been injected, (Tyndall effect) is a significant adverseevent experienced by some dermal filler patients. Tyndall effect is morecommon in patients treated for superficial fine line wrinkles.Embodiments of the present invention have been developed which providelong lasting, translucent fillers which can be injected superficially totreat fine lines and wrinkles, even in regions of relatively thin skin,without any resulting blue discoloration from Tyndall effect. Fine linesor superficial wrinkles are generally understood to be those wrinkles orcreases in skin that are typically found in regions of the face(forehead, lateral canthus, vermillion border/perioral lines) where theskin is thinnest, that is, the skin has a dermis thickness of less than1 mm. On the forehead the average dermal thickness is about 0.95 mm fornormal skin and about 0.81 mm for wrinkled skin. Dermis around thelateral canthus is even thinner (e.g. about 0.61 mm for normal skin andabout 0.41 mm for wrinkled skin). The average outer diameter of a 30 or32 gauge needle (needles that are typically used for fine line gelapplication) is about 0.30 and about 0.24 mm.

The present invention provides a dermal filler composition such asdescribed elsewhere herein, which does not result in Tyndall effect, ordoes not result in any visually perceptible blue discoloration resultingfrom Tyndall effect.

Methods of treating fine lines in the skin of a patient are alsoprovided. The methods generally comprise the steps of introducing intoskin of a patient, a composition such as described herein. For examplethe compositions comprise a mixture of a hyaluronic acid component, acrosslinking component crosslinking the hyaluronic acid, and an additiveother than the crosslinking component, the composition beingsubstantially optically transparent; and wherein the dermal fillercomposition exhibits reduced Tyndall effect relative to composition thatis substantially identical except without the additive.

In specific embodiments of the invention, gels are provided which arecrosslinked with HMDA and have a G′ of up to about 70 Pa, a G″/G′ aboveabout 0.65, an extrusion force of about 24 N or less, and a final HAconcentration of up to about 25 mg/ml.

In other embodiments, gels are provided which are crosslinked with 4 AA,and have a G′ of up to about 60 Pa, G″/G′ above about 0.70, an extrusionforce of about 30 N or less, and a final HA concentration of up to about24 mg/ml.

In yet other embodiments, gels are provided which are crosslinked withlysine methyl ester, and have a G′ of up to about 70 Pa, G″/G′ aboveabout 0.65, an extrusion force of about 24 N or less, and a final HAconcentration of up to about 25 mg/ml.

Many of the dermal filler gels in accordance with the invention containlidocaine, for example, at a lidocaine concentration of about 0.3 wt %.

These gels can be made as described herein, and are only provided asspecific examples of compositions in accordance with the invention.Further examples are provided below.

EXAMPLES Crosslinked HA gels via1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)Chemistry

Preparation of crosslinked HA-based gels, in accordance with certainembodiments of the invention are described in Examples 1 and 2 below. InExample 1, the gel is made via EDC chemistry using crosslinkerhexamethylene diamine (HMDA), and in Example 2, 3-[3-(3-aminopropoxy)-2,2-bis(3-amino-propoxymethyl)-propoxy]-propylamine (4 armamine-4 AA). Crosslinking is carried out under mild conditions, e.g.room temperature, for example, about 20 degrees C. to about 25 degreesC., and for example, at a low pH, for example, pH 5.4. The reactionsconditions are tuned to prepare highly reticulate gels with optimal gelproperties, excellent injectability and high final HA concentrations(˜24 mg/ml). It has been discovered by the inventors that it may beadvantageous to crosslink HA at very low hydration or reactionconcentrations, with a moderate amount of either HMDA or 4 AA, inconjunction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) or sulfonyl-NHS(sulfo-NHS), the coupling agents. The advantages may be that esterlinkage between HA may be reduced.

In an embodiment of the invention, a dermal filler composition fortreatment of wrinkles in skin is provided wherein the compositioncomprises a hyaluronic acid component crosslinked with 3-[3-(3-aminopropoxy)-2,2-bis(3-amino-propoxymethyl)-propoxy]-propylamine (4 AA) anda carbodiimide coupling agent.

Example 1 Formulation of HA-4 AA Hydrogel

13.33 g of 100 mM MES buffer pH 5.2 was added to a syringe containing400.2 mg of low molecular weight (LMW) HA. The LMW HA may have a meanmolecular weight of between about 310 KDa and about 840 KDa, forexample, less than about 600 KDa.

4 AA solution was prepared by dissolving 492.1 mg 4 AA in 999.2 mg of100 mM MES buffer pH 5.2, and adding 757 μl of 6 M HCl to bring the pHto 5.2.

EDC solution was prepared by dissolving 509.2 mg of EDC in 1156.4 mg 100mM MES buffer pH 5.2, and in a separate vial, 175 mg of NHS wasdissolved in 2635.8 mg of 100 mM MES buffer pH 5.2. Upon full hydrationof the HA, for about 1 hour, 99 μl of the 4 AA solution was added to thehydrated HA. The mixture was homogenized by 10 times syringe-to-syringemixing.

115 μl EDC solution, 115 μl NHS solution, and 140 μl of 100 mM MESbuffer pH 5.2 were then added to the homogenized paste and mixed 10times by syringe-to-syringe mixing.

The mixture was then transferred to a vial and crosslinked at roomtemperature for 5 hours, before the addition of 1.55 ml of 10×PBS bufferpH 7.4. The gel was allowed to swell for 3 days on a roller after whichit was forced through a 60 μm pore-size mesh.

The sized gel was transferred to a cellulose ester membrane dialysistubing MWCO 20 KDa and dialyzed against 1×PBS for 4 days, changing thebuffer twice a day.

The gel was dispensed in 1 ml COC syringes, centrifuged at 5000 RPM for5 min, and sterilized with moist steam. The gel had a final HAconcentration of 26 mg/ml.

In another embodiment of the invention, a dermal filler composition fortreatment of superficial wrinkles in skin is provided wherein thecomposition comprises a hyaluronic acid component crosslinked with alinear di-amine crosslinker, for example, hexamethylene diamine (HMDA)and a carbodiimide coupling agent. HMDA is the organic compound with theformula H₂N(CH₂)₆NH₂. The molecule is a diamine, consisting of ahexamethylene hydrocarbon chain terminated with amine functional groups.The dermal filler composition is administered into a dermal region of apatient to improve the appearance of skin.

Example 2 Formulation of a HA/HMDA Gel

20.0 g of 100 mM MES buffer pH 5.2 was added to a syringe containing1000.7 mg of LMW HA.

HMDA solution was prepared by dissolving 522 mg HMDA.HCl in 2008.5 mg of100 mM MES buffer pH 5.2, and adding 10 μl of 1 M NaOH to bring pH to5.2.

EDC solution was prepared by dissolving 508 mg of EDC in 1188.5 mg 100mM MES buffer pH 5.2, and in a separate vial, 44.2 mg of NHS wasdissolved in 1340.7 mg of 100 mM MES buffer pH 5.2. Upon full hydrationof the HA, for about 1 hour, 790 μl of the HMDA solution was added tothe hydrated HA. The mixture was homogenized by 10 timessyringe-to-syringe mixing.

490 μl EDC and 490 μl NHS solutions were then added to the homogenizedpaste. The mixture was again mixed 10 times by syringe-to-syringemixing, and then transferred to a vial and crosslinked at roomtemperature for 5 hours, before the addition of 18.0 ml of 1×PBS bufferpH 7.4.

The gel was allowed to swell for 3 days on a roller before it was forcethrough a 60 μm pore size mesh. The sized gel was placed in a celluloseester membrane dialysis tubing MWCO 20 KDa and dialyzed against 70% IPAfor 30 min, before the dialysis medium was changed to 1×PBS. The gel wasthen dialyzed for 4 days changing the buffer twice a day.

The gel was dispensed in 1 ml COO syringes, centrifuged at 5000 RPM for5 min to remove air bubbles, and sterilized with moist steam. The finalHA concentration of the gel was 25 mg/ml.

In yet another embodiment of the invention, a dermal filler compositionfor treatment of superficial wrinkles in skin is provided wherein thecomposition comprises a hyaluronic acid component crosslinked withlysine methyl ester and a carbodiimide coupling agent. Lysine is anessential amino acid and has chemical formula C₆H₁₄N₂O₂.

Example 3 Formulation of a HA/Lysine Hydrogel

400.5 mg of LMW HA was hydrated for 30 min. in a syringe by adding 8.0 gof 100 mM MES buffer pH 5.2. Lysine methyl ester (LME) solution wasprepared by dissolving 400.3 mg of LME.HCl in 1046.6 mg of 100 mM MESbuffer pH 5.2 in a vial. EDC solution was prepared by dissolving 288 mgof EDC in 1357.1 mg of 100 mM MES buffer pH 5.2 in a small vial, and NHSsolution was prepared by dissolving 61.0 mg of NHS in 962.8 mg of 100 mMMES buffer pH 5.2 in a separate vial. Upon complete hydration of HA, 261μl of LME solution was added to the hydrated HA. The mixture washomogenized by syringe-to-syringe mixing, 10 times, before adding 387 μlof EDC solution and 372 μl of NHS solution. The paste was again mixed 10times by syringe-to-syringe mixing. The mixture was then transferred toa vial and crosslinked at room temperature for 5 h. 6.21 ml of 1×PBSbuffer pH 7.4 was then added to the crosslinked gel. The gel was allowedto swell for 3 days on a roller before it was sized by forcing itthrough a 60 μm pore size stainless steel mesh. To convert thisHA-lysine methyl ester hydrogel to HA-lysine hydrogel, a small amount ofNaOH solution was added to the gel and mix to raise the gel pH to about12. The mixture was then placed in a water bath set at 50° C. for 5 minbefore the addition of hydrochloric acid to bring back the pH to ˜7.4.The gel was then placed in a cellulose ester membrane dialysis tubingMWCO 20 KDa and dialyzed against 70% IPA for 30 min, before the dialysismedium was changed to 1×PBS. The gel was dispensed in 1 ml COC syringes,centrifuged at 5000 RPM for 5 min to remove air bubbles, and sterilizedwith moist steam. The final HA concentration of the gel was 26 mg/ml.

Example 4 Determination of Rheological Properties of Gels of Examples1-3

An Oscillatory parallel plate rheometer, Anton Paar Physica MCR 301, wasused to measure the rheological properties of the gels. A plate diameterof 25 mm was used at a gap height of 1 mm. Measurements were done at aconstant temperature of 25° C. Each measurement consisted of a frequencysweep from 1 to 10 Hz at a constant strain of 2% and a logarithmicincrease of frequency followed by a strain sweep from 1 to 300% at aconstant frequency of 5 Hz with a logarithmic increase in strain. Thestorage modulus (G′) and the viscous modulus (G″) were obtained from thestrain sweep at 1% strain.

TABLE 2 Storage and viscous moduli of gels obtained from Example 1 toExample 3 Storage Modulus (G′) Viscous Modulus Sample ID Pa (G″) PaExample 1 263 49 Example 2 340 43 Example 3 560 90

Example 5 Extrusion Force Measurements of Gels of Examples 1-3

The force required to extrude the gels through a 30 gauge needle wasmeasured using an Instron 5564 and a Bluehill 2 software. The gels wereextruded from a 1 ml COO syringe through a 30 G½ TSK needle. The plungerwas pushed at a speed of 100 mm/min for 11.35 mm, and the extrusionforce was recorded.

TABLE 3 Extrusion force of gels obtained from Example 1 to Example 3Sample ID Extrusion force (N) Example 1 89 Example 2 32 Example 3 30

Example 6 Biocompatibility Testing of Gels of Examples 1-3

50 μl bolus injections of gel were implanted intradermally in the dorsalsurface of Sprague Dawley rats. The implants were removed at 1 week andanalyzed by histology with hematoxylin and eosin (H&E) staining, andCD68 staining which is a marker for mononuclear inflammation cells.Three 20× images of CD68 were scored from 0-4 based on the degree ofstaining. These values were then averaged out to give a sample score.Four samples were analyzed from each gel. Materials with score above 3.1are believed to be pro-inflammatory.

TABLE 4 Average CD68 scores of hydrogels from Examples 1 to 3 Sample IDAverage CD68 score Example 1 1.4 Example 2 2.4 Example 3 2.1

Example 7 Cytotoxicity Testing of Gels of Examples 1-3

In Vitro cytotoxicity tests of the gels were performed by NAMSAaccording to the Agarose Overlay Method of ISO 10993-5: biologicalEvaluation of Medical Devices—Part 5: Tests for In Vitro Cytotoxicity.Triplicate wells were dosed with 0.1 ml of test articles placed on afiltered disc, as well as 0.9% NaCl solution, 1 cm length of highdensity polyethylene as a negative control, and 1×1 cm² portion of latexas a positive control. Each was placed on an agarose surface directlyoverlaying a monolayer of L929 mouse fibroblast cells. After incubatingat 37° C. in 5% CO₂ for 24 h. The cultures were examined macroscopicallyand microscopically for any abnormal cell morphology and cell lysis. Thetest articles were scored from 0-4 based on the zone of lysis in theproximity of the samples. Test materials from Examples 1, 2, and 3scored 0 as test articles showed no evidence of causing any cell lysisor toxicity.

Example 8 Crosslinking HA with 4 AA Using EDC and NHS as Coupling Agents

32.55 g of 100 mM MES buffer pH 5.2 was added to a syringe containing1000.4 mg of LMW HA. 4 AA solution was prepared by dissolving 256.3 mg 4AA in 1039.8 mg of 100 mM MES buffer pH 5.2, and adding 380 μl of 6 MHCl to bring pH to 5.2. EDC solution was prepared by dissolving 251.2 mgof EDC in 1013.8 mg 100 mM MES buffer pH 5.2, and in a separate vial,74.7 mg of NHS was dissolved in 2020.0 mg of 100 mM MES buffer pH 5.2.Upon full hydration of the HA, ˜1 h, 260 μl of the 4 AA solution wasadded to the hydrated HA. The mixture was homogenized by 10 timessyringe-to-syringe mixing. 277 μl EDC and 273 μl NHS solutions were thenadded to the homogenized paste and again mix 10 times bysyringe-to-syringe mixing. The mixture was then transferred to a vialand crosslinked at room temperature for 5 h. before the addition of 6.4ml of 10×PBS buffer pH 7.4. The gel was allowed to swell for 3 days on aroller before it was force through a 60 μm pore size mesh. The sized gelwas placed in a cellulose ester membrane dialysis tubing MWCO 20 KDa anddialyzed against 1×PBS for 4 days changing the buffer twice a day. Thegel was dispensed in 1 ml COO syringes, centrifuge at 5000 RPM for 5min, and sterilized with moist steam. The gel had a final HAconcentration of 23 mg/ml.

Example 9 Crosslinking HA with HMDA Using EDC and NHS as Coupling Agents

20.0 g of 100 mM MES buffer pH 5.2 was added to a syringe containing1000.0 mg of LMW HA. HMDA solution was prepared by dissolving 260.9 mgHMDA hydrochloride in 2010.5 mg of 100 mM MES buffer pH 5.2, and adding2 μl of 1 M NaOH to bring pH to 5.2. EDC solution was prepared bydissolving 254.2 mg of EDC in 1188.4 mg 100 mM MES buffer pH 5.2, and ina separate vial, 44.3 mg of NHS was dissolved in 1341.8 mg of 100 mM MESbuffer pH 5.2. Upon full hydration of the HA, ˜1 h, 790 μl of the HMDAsolution was added to the hydrated HA. The mixture was homogenized by 10times syringe-to-syringe mixing. 490 μl EDC and 490 μl NHS solutionswere then added to the homogenized paste and again mixed 10 times bysyringe-to-syringe mixing. The mixture was then transferred to a vialand crosslinked at room temperature for 5 h. before the addition of 17.9ml of 1×PBS buffer pH 7.4. The gel was allowed to swell for 3 days on aroller before it was forced through a 60 μm pore size mesh. The sizedgel was placed in a cellulose ester membrane dialysis tubing MWCO 20 KDaand dialyzed against 1×PBS for 4 days changing the buffer twice a day.The gel was dispensed in 1 ml COC syringes, centrifuge at 5000 RPM for 5min, and sterilized with moist steam. The final HA concentration of thegel was 25 mg/ml

Example 10 HA-Lysine Hydrogels Using EDC and NHS as Coupling Agents

8.06 g of 100 mM MES buffer pH 5.2 was added to a syringe containing400.3 mg of LMW HA. Lysine methylester hydrochloride solution wasprepared by dissolving 400.3 mg lysine methylester hydrochloride in1046.6 mg of 100 mM MES buffer pH 5.2. EDC solution was prepared bydissolving 287.6 mg of EDC in 1364.6 mg 100 mM MES buffer pH 5.2, and ina separate vial, 60.2 mg of NHS was dissolved in 962.8 mg of 100 mM MESbuffer pH 5.2. Upon full hydration of the HA, ˜1 h, 132 μl of the lysinemethylester hydrochloride solution was added to the hydrated HA. Themixture was homogenized by 10 times syringe-to-syringe mixing. 196 μlEDC and 190 μl NHS solutions were then added to the homogenized pasteand again mixed 10 times by syringe-to-syringe mixing. The mixture wasthen transferred to a vial and crosslinked at room temperature for 5 h.before the addition of 6.67 ml of 1×PBS buffer pH 7.4. The gel wasallowed to swell for 3 days on a roller before it was forced through a60 μm pore size mesh. To convert HA-lysine methyl ester hydrogel toHA-lysine hydrogel, a small amount of NaOH solution was added to the geland mix to raise the gel pH to about 12. The mixture was then placed ina water bath set at 50° C. for 5 min before the addition of hydrochloricacid to bring back the pH to ˜7.4. The sized gel was placed in acellulose ester membrane dialysis tubing MWCO 20 KDa and dialyzedagainst 1×PBS for 4 days changing the buffer twice a day. The gel wasdispensed in 1 ml COO syringes, centrifuge at 5000 RPM for 5 min, andsterilized with moist steam. The gel had a final HA concentration of 26mg/ml.

Example 11 Gels Containing Lidocaine

1858 mg of lidocaine HCl was added into a 5 ml volumetric flask. Theflask was topped to the mark with 1×PBS buffer to give a lidocaine HClconcentration of 372 mg/ml. 200 μl of this lidocaine solution was thenadded to 24.5 mg of gel from Examples 8, 9 and 10 in a syringe. Themixture was forced to another syringe through a connector. This processwas repeated 20 times to obtain a homogenous mixture.

Example 12 Determination of Rheological Properties of Gels in Examples8-10

An Oscillatory parallel plate rheometer, Anton Paar Physica MCR 301, wasused to measure the rheological properties of the gels. A plate diameterof 25 mm was used at a gap height of 1 mm. Measurements were done at aconstant temperature of 25° C. Each measurement consisted of a frequencysweep from 1 to 10 Hz at a constant strain of 2% and a logarithmicincrease of frequency followed by a strain sweep from 1 to 300% at aconstant frequency of 5 Hz with a logarithmic increase in strain. Thestorage modulus (G′) and the viscous modulus (G″) were obtained from thestrain sweep at 1% strain.

TABLE 5 Storage and viscous moduli of gels obtained from Example 8 toExample 10 Storage Modulus (G′) Viscous Modulus Sample ID Pa (G″) PaExample 8 41 29.5 Example 9 67 42 Example 10 66 40

Example 13 Extrusion Force Measurements of Gels in Examples 8-10

The force required to extrude the gels through a 30 gauge needle wasmeasured using an Instron 5564 and a Bluehill 2 software. The gels wereextruded from a 1 ml COO syringe through a 30 G½ TSK needle. The plungerwas pushed at a speed of 100 mm/min for 11.35 mm, and the extrusionforce was recorded.

TABLE 6 Extrusion force of gels obtained from example 1 to example 4Sample ID Extrusion force (N) Example 8 19.5 Example 9 22 Example 10 22

Example 14 Cytotoxicity Testing of Gels, ISO 10993-5, Examples 8-10

In Vitro cytotoxicity tests of the gels were performed by NAMSAaccording to the Agarose Overlay Method of ISO 10993-5: biologicalEvaluation of Medical Devices—Part 5: Tests for In Vitro Cytotoxicity.Triplicate wells were dosed with 0.1 ml of test articles placed on afiltered disc, as well as 0.9% NaCl solution, 1 cm length of highdensity polyethylene as a negative control, and 1×1 cm² portion of latexas a positive control. Each was placed on an agarose surface directlyoverlaying a monolayer of L929 mouse fibroblast cells. After incubatingat 37° C. in 5% CO₂ for 24 h. the cultures were examined macroscopicallyand microscopically for any abnormal cell morphology and cell lysis. Thetest articles were scored from 0-4 based on the zone of lysis in theproximity of the samples. Test materials from examples 8, 9 and 10scored 0 as test articles showed no evidence of causing any cell lysisor toxicity.

Example 15 Tyndall Evaluation of Gels

In order to further support visual observations and carry outcomparative performance analysis of HA fillers, quantitative analysis ofTyndall effect was performed. Based on existing scientific understandingon light scattering and interaction of light with skin, two distinctapproaches based on (a) colorimetry, and (b) spectroscopy were employedto quantify Tyndall effect in skin. Based on these techniques threedistinct quantitative parameters (outlined below) were defined tomeasure Tyndall effect in vivo.

Tyndall Effect Visual Score:

The scale had a range of 1 to 5 with increments of 0.5. A score of 1 wasgiven to injection sites with normal skin tone and no bluediscoloration. A maximum score of 5 was given to thick and pronouncedblue discoloration (typically associated with Restylane or JuvédermUltra Plus). Three independent observers were trained on the scalebefore being blinded to score test samples.

Blue Component of Skin Color—“b”:

A chromameter (CM2600D, Konica Minolta, NJ) was used to quantify theblue color component of light remitted from skin sites injected with thevarious fillers. This was achieved by using the “b” component of L-a-bcolor scale.

“% Blue Light” Remitted from Skin:

A portable spectrophotometer (CM2600D, Konica Minolta, NJ) was used toquantify the % blue light remitted from skin in the total visible lightrange. This was achieved by integrating the area under the visible lightspectrum between 400-490 nm and normalizing it by the total area underthe spectrum (400-700 nm).

Gels of the present disclosure and commercial gels were injectedintradermally through a 27 G½ TSK needle using linear threadingtechnique into the thighs of two months old hairless rats. The gels wereimplanted superficially to mimic clinical fine line procedures. Testsfor Tyndall are performed 48 h after gel implantation. Before performingthe Tyndall tests, the animals are euthanized to improve contrast of theTyndall effect.

2 days after implantation of the gels it was found that gels of Examples8 to 10 exhibited no discernible Tyndall effect. Commercial fine linegels (Juvederm Refine and Restylane Touch, showed a marked bluishdiscoloration. (See FIG. 1).

A visual score of 1-5 with increments of 0.5, was used to score theinjection sites. Injection sites with score of 1 showed no skindiscoloration, while injections sites with score of 5 showed severe bluediscoloration of the skin. Spectroscopic analysis were also performed onthe injection sites with the aid of a chromatometer (CM2600D, KonicaMinolta, NJ). The blue component of skin color “b”, and the % of bluelight remitted from skin (400-700 nm) were independently measured. Gelsof Examples 8-10 exhibited no discernible Tyndall effect, and had lowervisual Tyndall score and % of blue light remitted values. The Tyndallscore and % of remitted blue light values were higher for the commercialgels.

Example 16 In Vivo Duration Evaluation of Gels, by MRI

150 μl of gel compositions of Examples 8 and 9, in accordance with theinvention, were injected intradermally on six different location on thedorsal side of female Sprague-Dawley rats; two contralateral sitescaudal to shoulder blades, two medial sites directly between caudal androstral sites, and two contralateral sites slightly rostral from knee,each location containing a different formulation. MRI scanning wasperformed with a 7 Tesla 70/30 Bruker Biospec instrument. Scanning wasperformed immediately after gel implantation (baseline), 12 weeks aftergel implantation, and 24 weeks after gel implantation. In vivodegradation and gel stability was determined by calculating the volume,surface area, and surface area to volume ratio at each time point.

As shown, the present gels show substantial improvement in longevity invivo when compared to Commercial Fine Line Filler III (Belotero Soft), acommercial HA-based dermal filler that is marketed as being useful fortreating superficial wrinkles in skin.

Example 17 Compositions of the Invention for Treatment of PeriorbitalLines

A 40 year old thin woman presents with fine wrinkles in the periorbitalregion and requests dermal filler treatment. Using a 30 gauge needle,the physician introduces 0.6 ml of a HA-based gel in accordance with theinvention (such as that described in Example 8) superficially into thefine lines beneath her eyes and in the tear trough region using linearthreading technique. Although the gel is introduced superficially, noblue discoloration is observed and the patient is satisfied with theresults.

Example 18 Compositions of the Invention for Treatment of PeriorbitalLines

A 35 year old man having Fitzpatrick skin type III presents with finewrinkles in the glabellar region and requests dermal filler treatment.Using a 27 gauge needle, the physician introduces 1.0 ml of a HA-basedgel in accordance with the invention (such as that described in Example9) superficially, about 0.8 mm deep, into the glabellar lines usingconventional technique. Although the gel is introduced, no bluediscoloration is observed. Two weeks after treatment, there is still nobluish discoloration. The patient reports he is satisfied with theresults.

In closing, it is to be understood that although aspects of the presentspecification have been described with reference to the variousembodiments, one skilled in the art will readily appreciate that thespecific examples disclosed are only illustrative of the principles ofthe subject matter disclosed herein. Therefore, it should be understoodthat the disclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,those skilled in the art could make numerous and various modificationsor changes to or alternative configurations of the disclosed subjectmatter can be made in accordance with the teachings herein withoutdeparting from the spirit of the present specification. Changes indetail may be made without departing from the spirit of the invention asdefined in the appended claims. Lastly, the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. In addition, it is intended that all mattercontained in the above description or shown in the accompanying drawingsshall be interpreted as illustrative only and not limiting. Accordingly,the present invention is not limited to that precisely as shown anddescribed.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” As used herein,the term “about” means that the item, parameter or term so qualifiedencompasses a range of plus or minus ten percent above and below thevalue of the stated item, parameter or term. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thespecification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the invention are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents arebased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

What is claimed is:
 1. A method of making an injectable dermal fillercomposition for treating wrinkles in skin, the method comprising thesteps of crosslinking hyaluronic acid (HA) with a di-amine or multiaminecrosslinker in the presence of a carbodiimide coupling agent.
 2. Themethod of claim 1 wherein the crosslinker is a linear di-aminecrosslinker.
 3. The method of claim 2 wherein the crosslinker ishexamethylene di-amine (HMDA).
 4. The method of claim 1 wherein thecrosslinker is 3-[3-(3-aminopropoxy)-2,2-bis(3-amino-propoxymethyl)-propoxy]-propylamine (4 AA). 5.The method of claim 1 wherein the crosslinker is made up of at leastthree polyethylene glycol (PEG) chains and no more than eight PEG chainsemanating from a central point.
 6. The method of claim 1 wherein thecrosslinker is made up of at least three polyethylene glycol (PEG)chains and no more than eight PEG chains emanating from a central point,each chain having a terminal amine group.
 7. The method of claim 1wherein the crosslinker contains polyethylene glycol (PEG) chains havinga least one ethylene glycol unit and not more than 55 ethylene glycolunits.
 8. The method of claim 1 wherein the crosslinker is lysine methylester.
 9. The method of claim 1 wherein the coupling agent is1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC). 10.The method of claim 1 wherein the crosslinking takes place in thepresence of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride(EDC).
 11. The method of claim 10 wherein the crosslinking takes placein the presence of N-hydroxysuccinimide (NHS) orN-hydroxysulfosuccinimide (sulfoNHS).
 12. A dermal filler compositionfor treatment of superficial wrinkles in skin, the compositioncomprising: a hyaluronic acid component crosslinked with a di-amine ormultiamine crosslinker; and a carbodiimide coupling agent; thecomposition being substantially optically transparent; and thecomposition exhibiting reduced or no perceptible blue discoloration whenadministered into a dermal region of a patient.
 13. The composition ofclaim 12 wherein the dermal region is a tear trough region, a glabellarline, a periorbital region or a forehead region.
 14. The composition ofclaim 12 which exhibits reduced or no perceptible blue discolorationwhen administered at a depth of no greater than about 1.0 mm into thedermal region.
 15. The composition of claim 12 which exhibits reduced orno perceptible blue discoloration when administered at a depth of nogreater than about 0.8 mm into the dermal region.
 16. The composition ofclaim 12 which exhibits reduced or no perceptible blue discolorationwhen administered at a depth of no greater than about 0.6 mm into thedermal region.
 17. The composition of claim 12 wherein the crosslinkeris a linear di-amine crosslinker.
 18. The composition of claim 12wherein the crosslinker is hexamethylene di-amine (HMDA).
 19. Thecomposition of claim 12 wherein the crosslinker is 3-[3-(3-aminopropoxy)-2,2-bis(3-amino-propoxymethyl)-propoxy]-propylamine (4 AA). 20.The composition of claim 12 wherein the crosslinker is present in thecomposition at a concentration between about 10 μM to about 50 μM. 21.The composition of claim 12 wherein the crosslinker is made up of atleast three polyethylene glycol (PEG) chains and no more than eight PEGchains.
 22. The composition of claim 12 wherein the crosslinker is madeup of at least three polyethylene glycol (PEG) chains and no more thaneight PEG chains emanating from a central point.
 23. The composition ofclaim 12 wherein the crosslinker is made up of at least threepolyethylene glycol (PEG) chains and no more than eight PEG chainsemanating from a central point, each chain having a terminal aminegroup.
 24. The composition of claim 12 wherein the crosslinker containspolyethylene glycol (PEG) chains having a least one ethylene glycol unitand not more than 55 ethylene glycol units.
 25. The composition of claim12 wherein the crosslinker is lysine methyl ester.
 26. The compositionof claim 12 wherein the coupling agent is a water soluble carbodiimide.27. The composition of claim 12 wherein the coupling agent is1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC). 28.The composition of claim 12 wherein the coupling agent is present in thecomposition at a concentration between about 20 μM to 100 μM.
 29. Amethod of treating fine lines in skin comprising administering a dermalfiller composition made by a method of claim 12 into a dermal region ofa patient.